Identification of a novel Drosophila opsin reveals specific patterning of the R7 and R8 photoreceptor cells.

The function of the compound eye is dependent upon a developmental program that specifies different cell fates and directs the expression of spectrally distinct opsins in different photoreceptor cells. Rh5 is a novel Drosophila opsin gene that encodes a biologically active visual pigment that is expressed in a subset of R8 photoreceptor cells. Rh5 expression in the R8 cell of an individual ommatidium is strictly coordinated with the expression of Rh3, in the overlying R7 cell. In sevenless mutant files, which lack R7 photoreceptor cells, the expression of the Rh5 protein in R8 cells is disrupted, providing evidence for a specific developmental signal between the R7 and R8 cells that is responsible for the paired expression of opsin genes.

Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia.

Vascular endothelial growth factor-B (VEGF-B) is closely related to VEGF-A, an effector of blood vessel growth during development and disease and a strong candidate for angiogenic therapies. To further study the in vivo function of VEGF-B, we have generated Vegfb knockout mice (Vegfb(-/-)). Unlike Vegfa knockout mice, which die during embryogenesis, Vegfb(-/-) mice are healthy and fertile. Despite appearing overtly normal, Vegfb(-/-) hearts are reduced in size and display vascular dysfunction after coronary occlusion and impaired recovery from experimentally induced myocardial ischemia. These findings reveal a role for VEGF-B in the development or function of coronary vasculature and suggest potential clinical use in therapeutic angiogenesis.

HET/SAF-B overexpression causes growth arrest and multinuclearity and is associated with aneuploidy in human breast cancer.

HET/SAF-B was originally cloned as a nuclear matrix protein that bound to matrix attachment regions and as a transcriptional repressor of the small heat shock protein hsp27. In addition, we have found recently that HET/SAF-B is also a corepressor of estrogen receptor activity. Estrogen receptor has a very well-described role in breast cancer, and aberrant expression of nuclear matrix and heat shock proteins has also been implicated in breast tumorigenesis. Therefore, we asked whether HET/SAF-B itself could be important in breast cancer. Toward this goal we examined its expression in breast cancer cell lines and asked whether HET/SAF-B can affect breast cancer cell proliferation. Finally, we studied HET/SAF-B expression in clinical breast cancer samples. HET/SAF-B protein and mRNA were detected at varying levels in all of the eight breast cancer cell lines examined. Using a number of different approaches to modulate the level of HET/SAF-B protein in the cell, we found that HET/SAF-B levels are inversely correlated with cell proliferation. In addition,transfection of HET/SAF-B fused to the green fluorescent protein led to the formation of multinucleated cells not observed in cells transfected with green fluorescent protein alone, suggesting that this effect is a direct result of HET/SAF-B overexpression. Western blot analysis of HET/SAF-B in 61 human breast tumors revealed widely varying levels of HET/SAF-B expression, with some tumors (16%) lacking any detectable HET/SAF-B. Statistical analysis showed that high HET/SAF-B expression in these tumors was associated with low S-phase fraction and with aneuploidy, consistent with our results from transfection experiments in tissue culture cells. We conclude that HET/SAF-B plays an important role in breast cancer, and we discuss possible mechanisms of the involvement of HET/SAF-B in cell proliferation and division.

Estrogen receptor corepressors -- a role in human breast cancer?

Estrogen receptor alpha (ERalpha) has an established role in promoting breast cancer. Transcriptional activation by ERalpha is a complex and multistep process, and it is influenced by coactivator and corepressor proteins that can either positively or negatively modulate ERalpha-mediated transcriptional activity. Corepressors are proposed to provide a counterbalance to the estrogen-induced transactivation, and represent a potential mechanism employed by the cell to regulate hormonal responses. In this review, we present evidence from tissue culture, animal and clinical studies, supporting the hypothesis that corepressors are crucial regulators of ERalpha-mediated action, and that their loss could promote breast cancer development and resistance to endocrine therapy. We propose that ERalpha corepressors play an important biological role by controlling the magnitude of the estrogen response, mediating antiestrogen inhibition of ERalpha, repressing DNA-bound ERalpha in the absence of the ligand, and conferring active repression of ERalpha-downregulated genes. Different ERalpha corepressors regulate steroid receptor activity through a variety of mechanisms, including formation of multiprotein complexes that are able to affect chromatin remodeling, histone deacetylation, or basal transcription. Other mechanisms include competition with coactivators, interference with DNA binding and ERalpha homodimerization, alteration of ERalpha stability, sequestration of ERalpha in the cytoplasm, and effects on RNA processing. Most ERalpha corepressors can control the receptor's activity through more than one mechanism, and it is possible that the synergy between different pathways cooperates to fully inhibit ERalpha transcriptional activity, and create an integrated response to a variety of different cellular signaling pathways. We will discuss the role of corepressors in tumor suppression and the link they might present between ERalpha regulation and DNA repair. Finally, we will discuss major challenges in the field and speculate on the exciting findings that await us in the next few years.

Antiparasitic properties of medicinal plants and other naturally occurring products.

Parasitic diseases remain a major public health problem affecting hundreds of millions of people, particularly in tropical developing countries. The limited availability and affordability of pharmaceutical medicines means that the majority of the world's population depends on traditional medical remedies, and it is estimated that some 20,000 species of higher plant are used medicinally throughout the world. Many well-known drugs listed in the modern pharmacopoeia have their origins in nature, including, for example, quinine from the bark of the Cinchona tree for the treatment of malaria, which has been followed by the subsequent development of the synthetic derivatives chloroquine, amodiaquine, primaquine and mefloquine. More recently, the wider recognition of the antimalarial activity of artemisinin from the herb Artemisia annua has led current research to focus on the development of a large number of synthetic and semisynthetic compounds, which are more active than artemisinin. There is an increasing awareness of the potential of natural products, which may lead to the development of much-needed new antiparasitic drugs. In this chapter, we have drawn together a comprehensive list of medicinal plants and other natural products that have been shown to have activity against human and, to a lesser extent, animal parasites. In addition, some of the opportunities and difficulties in working with natural products have been reviewed and discussed, including the problems involved with evaluating complex mixtures of compounds which may occur in extracts, problems associated with differentiating between general cytotoxicity and genuine antiparasitic activity, and the hope that new technologies will rapidly accelerate new drug discovery and development in this field. Nevertheless, the way forward for natural product medicines, including the conservation of recognized natural products and protection of general biodiversity, the discovery and development process, and the promotion and usage of existing remedies, presents some difficult challenges. Following an initiative by the World Health Organization in August 2000, there is now the opportunity to evaluate scientifically many more traditional medicines and other natural products in validated antiparasite and toxicity screens, which will help establish which substances have potential for new pharmaceutical products. The use of 'untested' traditional medicines will no doubt continue, and there is an urgent need to distinguish between the efficacious and safe products and the ineffective and/or unsafe products, particularly since many remedies are being more widely promoted in developing countries.

Evidence for the occurrence of respiratory electron transport in adult Brugia pahangi and Dipetalonema viteae.

Mixed-sex adult stages of Brugia pahangi and Dipetalonema viteae, in the absence of exogenous substrate, consumed oxygen at rates of 4.18 +/- 0.38 and 2.12 +/- 0.20 ngatoms O2 min-1 mg-1 dry wt. respectively. When calculated on a unit dry weight basis the endogenous O2 consumption rates (E-QO2) of mature adult male macrofilariae of B. pahangi and D. viteae were significantly greater than those of mature females, although the E-QO2 calculated per individual worm was essentially similar irrespective of sex. When assayed as separate unisexual groups, the oxygen uptake of male and female macrofilariae of both species was inhibited by classical inhibitors of respiratory electron transport (RET), and showed classical substrate bypass phenomena in response to succinate and ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine with respect to the RET inhibitors rotenone (inhibitor of complex I) and antimycin A (inhibitor of complex III). Since male worms elicited similar responses to the classical RET inhibitors as did mixed-sex and/or adult female populations, the possibility that developmental stages contained within the female filariids were contributing in any significant manner to the overall responses observed with the RET inhibitors can be discounted. Such responses as observed with live-intact macrofilariae are normally elicited only by mitochondrial preparations and suggest that the cuticles of both species are permeable to rotenone, succinate, antimycin A, N,N,N',N'-tetramethyl-p-phenylenediamine, azide and cyanide. The uncoupler 2,4-dinitrophenol stimulated the endogenous rate of oxygen consumption (E-QO2) of intact B. pahangi at 33-160 microM, indicating the probable occurrence of RET-coupled oxidative phosphorylation. Higher concentrations of 2,4-dinitrophenol proved inhibitory. Respiratory studies on subcellular fractions substantiated the responses elicited by the intact parasites, suggesting the presence of antimycin A-sensitive and -insensitive RET pathways capable of utilising alpha-glycerophosphate, succinate, and malate as substrates. Both B. pahangi and D. viteae macrofilariae therefore probably possess branched RET-pathways bifurcating on the substrate side of RET-complex III. The rates of substrate oxidation in terms of QO2 mg-1 mitochondrial protein compare well with those observed with other nematode parasites.

Characterisation and purification of pyruvate:ferredoxin oxidoreductase from Giardia duodenalis.

The major 2-oxoacid oxidoreductase (2-OR), pyruvate:ferredoxin oxidoreductase (PFOR) from Giardia duodenalis has been purified to apparent homogeneity. A second 2-OR with a preference for alpha-ketobutyrate as substrate was identified and was removed from PFOR containing fractions during purification. Only PFOR and the second 2-OR were identified in gels of crude Giardia extracts assayed for 2-OR activity. The native form of PFOR which is membrane associated, is a homodimer of 138 kDa subunits. Pyruvate is the preferred substrate: alpha-ketobutyrate and oxaloacetate, but not phenyl-pyruvate or alpha-ketoglutarate, are decarboxylated. PFOR from Giardia is more stable than PFOR from most other organisms and purified PFOR can be stored without deterioration at -70 degrees C. Purified PFOR donates electrons to Giardia ferredoxin (Fd I) with concomitant reduction of metronidazole. However, two other Giardia ferredoxins did not accept electrons from PFOR. Consistent with the involvement of PFOR in metronidazole activation, the activity of pyruvate dependent 2-OR activity was decreased in all metronidazole-resistant lines tested but not in furazolidone-resistant lines. The presence of three different ferredoxins and two 2-ORs in Giardia suggests that a number of different electron transport pathways operate in this organism providing unusual metabolic flexibility for a eukaryote.

Developmentally regulated expression and secretion of a polymorphic antigen by Onchocerca infective-stage larvae.

In order to analyse the developmental biology of Onchocerca spp. with a view to identifying molecules with specialised functions, we have devised a novel method for labelling proteins synthesised by larvae during growth in the vectors. Pulse labelling of Onchocerca lienalis by micro-injections of [35S]methionine into blackflies have revealed a major acidic protein of 23 kDa which is developmentally expressed almost exclusively by infective, third-stage larvae. The protein appears to be antigenically conserved between O. lienalis and Onchocerca volvulus, but exhibits size polymorphisms both among species and among individual organisms. It continues to be elaborated after terminal differentiation of the parasite in flies, but not by post-infective larvae entering the phase of development in the vertebrate host. A shift in temperature from 26 degrees C to 37 degrees C triggers secretion of the 23-kDa molecule as a discrete event 24-72 h after transmission. The labelling technique has been successfully employed with filarial species that develop in mosquitoes, and in principle should be widely applicable to the study of endoparasite gene expression within arthropods.

2-acetylpyridine thiosemicarbazones. 13. Derivatives with antifilarial activity.

Several members of a series of 2-acetylpyridine thiosemicarbazones possess in vivo and in vitro macrofilaricidal properties. The most promising of the group tested is N4-(2-aminophenyl)-2-[1-(2-pyridinyl)ethylidene]-hydrazinecarbothioam ide (4), which suppressed 100% of the macrofilariae of Brugia pahangi and 94% of those of Acanthocheilonema viteae in the jird at a dose of 25 mg/kg per day x 5. Compounds 4 and 14 were also shown to inactivate or kill Onchocerca gutturosa and Onchocerca volvulus adult worms as measured by the loss of their motility or the inhibition of the conversion by the worms of the dye MTT to formazan.

In vitro cultivation and development of Onchocerca volvulus and Onchocerca lienalis microfilariae.

Onchocerca volvulus and O. lienalis skin-derived microfilariae (mf) were cultured in vitro; parasite viability was assessed at intervals by measuring their ability to develop in Simulium ornatum. In the presence of monkey kidney feeder cells, both species retained full viability when cultured for up to 5 hr before intrathoracic injection into Simulium. In the absence of feeder cells and in contrast to O. lienalis, O. volvulus mf rapidly lost their viability. In further trials (including feeder cells), O. volvulus mf retained full viability for 14 days, while O. lienalis mf retained full viability for a least 19 days but with a proportion able to develop to third-stage larvae (L3) after 70 days in culture. In experiments using this system to culture O. volvulus mf (ex utero) derived from adult female worms but with the addition of reduced glutathione and/or 20-hydroxyecdysone, parasites were consistently more active over a 70-day period than those cultured without these additives. None of the mf cultured without additives were able to develop to L3 in Simulium when tested for up to 51 days in culture, while a proportion of mf incubated with reduced glutathione and/or 20-hydroxyecdysone produced small but significant numbers of L3 after 28, 43, and 51 days, representing the first time that uterine mf have been cultured to a form infective for the vector.

Improved development of Brugia microfilariae following cryopreservation in liquid nitrogen using a technique suitable for field conditions.

A technique for improved cryopreservation at -196 degrees C of Brugia spp. microfilariae has been developed by modifications of a procedure previously used with Onchocerca spp. A double incubation in ethanediol (ED) solutions, firstly at 37 degrees C in 10% (v/v) ED for 15 min and secondly at 0 degrees C in 40% (v/v) ED for 45 sec followed by plunging into liquid nitrogen, resulted in over 90% of the microfilariae of B. malayi exhibiting normal motility. When used with B. pahangi microfilariae, followed by blood-feeding to Aedes aegypti (Bels New strain) a third-stage larvae yield of 4.04 per fly was obtained, compared with 5.16 in the unfrozen group. This represents an infectivity level of 78% compared with unfrozen controls. The technique also has the benefits of being suitable for field conditions, requiring no sophisticated equipment, merely some ice, ethanediol and liquid nitrogen.

Comparison of the sensitivity of different geographical isolates of Onchocerca volvulus microfilariae to ivermectin: effects of exposure to drug on development in the blackfly Simulium ornatum.

Four geographical isolates (Ghana forest, Ghana savannah, Cameroon forest, Guatemala) of Onchocerca volvulus microfilariae (mf) and O. lienalis mf (UK) were examined for their sensitivity to ivermectin by incubation in vitro in drug followed by assessing their ability to develop in the blackfly Simulium ornatum after intrathoracic injection. Parasites were incubated for 30 min in ivermectin (10(-6) to 10(-9) M), which resulted in a concentration dependent decrease in the numbers of parasites surviving and developing in the insect; there were significant reductions in parasite recoveries from all isolates in the 10(-6) M to 5 x 10(-8) M ivermectin groups, but no significant effect was seen following incubation in concentrations of 10(-8) M and below. Experiments consistently demonstrated that the 4 isolates of O. volvulus were similarly sensitive to ivermectin (in the 10(-7) M ivermectin groups there was a reduction of 76.3% to 85.1% in numbers of infective larvae, and 60.9% to 85.5% in numbers of all larval stages, compared to controls); O. lienalis mf were significantly more sensitive (100% reduction in infective larvae, 98.7% reduction in all larval stages). This baseline information on drug sensitivity and techniques should prove useful for examining populations of O. volvulus for possible development of drug resistance in the future.

The effects of ivermectin used in combination with other known antiparasitic drugs on adult Onchocerca gutturosa and O. volvulus in vitro.

The effects of ivermectin at a concentration of 3.13 x 10(-6) M used in combination with other antiparasitic drugs on the viability of adult Onchocerca in vitro were assessed using MTT colorimetry and worm motility levels. When ivermectin was used against male O. gutturosa over a 7 d period in combination with suramin (5 x 10(-5) M), CGP 6140 (3.13 x 10(-6) M), CGP 20376 (1.95 x 10(-7) M), mefloquine (3.13 x 10(-6) M), levamisole (3.13 x 10(-6) M), mebendazole (5 x 10(-5) M), flubendazole (5 x 10(-5) M) and albendazole (5 x 10(-5) M), there was either no increased effect or only a marginally increased effect on motility levels when compared with the use of ivermectin alone. MTT colorimetry revealed that in most cases there was a cumulative effect of the 2 drugs used in combination but not a synergistic effect. In a trial extended to 26 d it was demonstrated that the combination of ivermectin and suramin did not produce a greater inhibition of motility than ivermectin alone. Using female O. volvulus, the activity of ivermectin, CGP 6140 and the 2 drugs combined was examined. The motility of all 3 groups exposed to drug(s) was suppressed by 24 h compared with controls. MTT colorimetry performed on day 7, using the pre-weighed anterior end of each worm, illustrated that ivermectin alone produced a 43.4% inhibition of formazan formation compared with controls, CGP 6140 alone produced 50.6% inhibition, while the drug combination produced a 72% inhibition, equivalent to the heat-killed control.(ABSTRACT TRUNCATED AT 250 WORDS)

Onchocerca gutturosa and O. volvulus: studies on the viability and drug responses of cryopreserved adult worms in vitro.

The viability and drug responses of cryopreserved adult Onchocerca have been examined in vitro. Male worms were cryopreserved in liquid nitrogen (-196 degrees C) using ethanediol as a cryoprotectant in a 2-step incubation procedure. After thawing, 85-90% of O. gutturosa males were normally motile. These motile worms were evaluated for viability using 4 measurements (long-term motility/survival in culture; [U-14C]adenine uptake and leakage; glucose utilization; MTT-formazan colorimetry) and were no different from unfrozen controls. Subsequent experiments demonstrated that the motility responses of cryopreserved worms exposed to the antifilarial drugs ivermectin, CGP 6140 and levamisole were virtually identical to unfrozen controls. Some success was also obtained with this technique in cryopreserving O. volvulus males, with 2 thawed specimens surviving in culture for 93 and 106 d respectively. Following collagenase isolation, female worms were cryopreserved in medium +10% serum without protectant at -79 degrees C. A batch of 8 female O. gutturosa were all motile when thawed 14 d later, with a mean survival time (based on 5 specimens) of 71 d (range 60-90). However, a batch of worms transferred from -79 degrees C to -196 degrees C were badly damaged when thawed. Female O. volvulus were cryopreserved at -79 degrees C in Guatemala and sent by air freight on solid CO2 to the UK. Most specimens were active when thawed. Survival of motile specimens ranged from 7 to 272 d in culture. It is concluded that these techniques are of practical value for the storage and transportation of adult Onchocerca.

Comparison of the sensitivity of different geographical races of Onchocerca volvulus microfilariae to ivermectin: studies in vitro.

This study was designed to provide baseline information on the sensitivity of 4 geographical isolates of Onchocerca volvulus microfilariae (mf) (Ghana forest, Ghana savanna, Cameroon forest and Guatemala) to ivermectin, and to develop an in vitro system with which to examine parasites for the possible development of drug resistance. Drug effects were best visualized in the presence of monkey kidney (LLCMK2) feeder cells in the culture system (MEM medium+20% serum), since mf maintained in the absence of cells declined in condition rapidly. Incubation of Ghana forest mf (+cells) in ivermectin (10(-5)-10(-10) M) caused a decrease in motility index (MI) scores in a concentration-dependent fashion; drug effects could be observed as early as 6 h, but cultures maintained for up to 8 d showed greater differences between control and drug groups with increasing time. All 4 O. volvulus isolates and O. lienalis (bovine) were compared for their response to ivermectin (10(-7) M): O. lienalis mf were significantly more sensitive (78%) reduction in MI scores on day 8) than the O. volvulus isolates (33.4-47.7% reduction). O. volvulus microfilariae ex utero generally displayed lower levels of motility and were slightly less inhibited by ivermectin than were skin mf. The in vitro system described can distinguish between the populations of mf studied on the basis of differing MI responses to ivermectin and, when combined with assays to test the infectivity of mf to blackflies following exposure to drug, will provide methods with which to examine parasites for the possible development of resistance.

Resistance to the nitroheterocyclic drugs.

The nitroheterocyclic drugs have been available since the early 1960's for the treatment of anaerobic protozoa. The application of these drugs has widened since then and they are presently used to treat anaerobic pathogenic bacteria and protozoa. The activity of the nitroheterocyclic drugs depends on the all-important nitro group attached to the imidazole or furan ring. Although the nitro radicals, generated by reduction of the parent drugs, are similar for both families of nitroheterocyclics, the nitroimidazoles and the nitrofurans, the electron potential of each is different and thus the mechanism of action depends on different pathways. The nitroimidazoles depend on reduction by ferredoxin or flavodoxin. The nitrofurans require nitroreductase activity, but the natural substrate of these enzymes has not been identified. Increased use of nitroheterocyclic drugs, in response to drug resistance to other commonly used antibiotics, has in turn resulted in drug resistance to a number of nitroheterocyclic drugs. Bacteroides strains and other bacteria, including Helicobacter, have developed resistance. Among the protozoa, Trichomonas has developed resistance to metronidazole via a number of mechanisms, especially a decrease in drug reduction, as a result of alterations in the electron transport pathways. Resistance to both types of nitroheterocyclic drugs has been reported in Giardia. Although resistance to these drugs is not widespread, their increased use world-wide as a prophylaxis and in chemotherapy will inevitably result in increased resistance in organisms commonly found in asymptomatic infections, including Trichomonas, Giardia and Entamoeba. However, the variety of substitutions which can be attached to the ring structures has led to a great variety of drugs being synthesised, some of which are many-fold more active than the commonly prescribed nitroheterocyclics. With careful administration of currently available drugs and continued interest in synthesising more active compounds, we can optimistically expect to have useful nitroheterocyclic drugs available for some time.

Potential transition state phosphoramidate inhibitors of beta-tubulin as antifilarial agents.

Transition state phosphoramidate inhibitors of beta-tubulin were designed as potential antifilarial agents. The reaction of 2-aminobenzimidazole with diisopropyl phosphite and carbon tetrachloride at a low temperature gave the unexpected 1-diisopropoxyphosphoryl-2-aminobenzimidazole, which on heating gave the novel benzimidazole derivative, 2-(diisopropoxyphosphoryl)aminobenzimidazole. Both products were fully characterized and the synthetic procedure to both compounds was optimized. The procedure was used to prepare the related 5-benzoyl-2-(diisopropoxyphosphoryl)aminobenzimidazole and 5-benzoyl-2-(diethoxyphosphoryl)aminobenzimidazole (1d). In a preliminary trial against Brugia pahangi compound 1d was found to have no antifilarial activity. This lack of activity may be attributed to its extreme insolubility and thus low bioavailability. The synthesis of analogous, more soluble, phosphorothioate-substituted benzimidazoles using the same methods may yield compounds with greater antifilarial activity.

The development of a laboratory model for onchocerciasis using Onchocerca gutturosa: in vitro culture, collagenase effects, drug studies and cryopreservation.

A range of culture conditions were examined to optimize parasite maintenance. Using male worms in a cell-free system, good results were obtained with medium NCTC 135 + 10% inactivated calf serum (IFCS) in an atmosphere of 95% N2/5% CO2 (median survival time 45 days). Survival was increased to 6-7 months using medium MEM + 10% IFCS + LLCMK2 (monkey kidney) feeder cells in a gas phase of 5% CO2 in air. Worms exposed to collagenase solution (5 mg/ml) were subsequently less motile and survived shorter periods compared to unexposed controls. The drug responses of worms (in vitro) were examined using 13 antiparasitic compounds. Ivermectin and CGP 6140 were among the most active, with the majority of drugs significantly affecting motility levels at a concentration of 5 x 10(-5) M or less. This system may provide useful information on the intrinsic activity of new compounds. A technique was developed for the successful cryopreservation of males in liquid nitrogen using ethanediol as a cryoprotectant in a 2-step incubation procedure, thereby enabling the long-term storage and transportation of worms. In conclusion, the common bovine parasite O. gutturosa provides a practical alternative for research in the absence of O. volvulus.

The effects of ivermectin on the viability of Onchocerca lienalis microfilariae in vitro and on their subsequent development in the blackfly vector, Simulium ornatum.

Experiments examined the sensitivity of Onchocerca lienalis microfilariae (mf) to ivermectin in vitro using a range of parameters to measure viability. Incubation in drug (1 x 10(-4) and 5.71 x 10(-8) M) caused an immediate decrease in motility levels in a concentration dependent fashion with a continuing decline over 24 h; the inclusion of a monkey kidney cell (LLCMK2) feeder layer in the culture system partially abrogated drug effects. The use of feeder cells allowed the longer-term culture of mf; incubation in drug at low concentrations (5.71 x 10(-8) and 1 x 10(-10) M) significantly reduced motility levels during a 17 day trial. Using MTT colorimetry it was found that 10,000 untreated mf per replicate sample were required to produce a sufficient quantity of formazan to make comparisons, and that formazan formation was most rapid during the first 30 min of incubation in MTT but continued up to 3-4 h. Following incubation of mf in ivermectin (1 x 10(-4) and 5.71 x 10(-8) M) for 24 h, only the higher concentration inhibited formazan formation (51%), while motility levels were reduced by 94% and 45% respectively. Using the same drug concentrations, mf viability was assessed by measuring the uptake of [3H]2-deoxy-D-glucose; uptake was only significantly reduced (40%) by the higher concentration, while motility levels were reduced by 77% and 63% respectively.(ABSTRACT TRUNCATED AT 250 WORDS)