A novel assay for the detection of anthelmintic activity mediated by cuticular damage to nematodes: validation on Caenorhabditis elegans exposed to cysteine proteinases.

Plant cysteine proteinases (CPs) from Carica papaya kill parasitic and free-living nematodes in vitro by hydrolysis of the worm cuticle, a mechanism that is different to all commercially available synthetic anthelmintics. We have developed a cheap and effective, rapid-throughput Caenorhabditis elegans-based assay for screening plant CP extracts for anthelmintic activity targeting cuticular integrity. The assay exploits colorimetric methodology for assessment of cuticular damage, and is based on the ability of viable cells to incorporate and bind Neutral red dye within lysosomes and to release the dye when damaged. Living worms are pre-stained with the dye, exposed to CPs and then leakage of the dye through the damaged cuticle is quantified by spectrophotometry. In contrast to motility assays and semi-subjective interpretation of microscopical images, this colorimetric assay is independent of observer bias. Our assay was applied to a series of C. elegans bus mutant strains with leaky cuticles and to cystatin knockout mutants. At ambient temperature and over 0.5-24 h, both bus mutants and the cystatin knockouts were highly susceptible to CPs, whereas wild-type Bristol N2 worms were essentially unstained by Neutral red and unaffected by CPs, providing validation for the utility of this assay.

Toxicity of the dithiocarbamate fungicide mancozeb to the nontarget soil nematode, Caenorhabditis elegans.

We have previously shown that the dithiocarbamate fungicide, Mancozeb, strongly induces lacZ reporter expression from an endogenous heat-shock promoter (hsp16) in the PC72 transgenic strain of the nematode Caenorhabditis elegans. Such evidence of organismal stress, in a nontarget species at subapplication concentrations, was much less apparent for the related fungicide, Maneb, which only weakly induced reporter expression. We now show that reporter induction by Mancozeb is marginal (<60%) after a few hours' exposure, but increases substantially (to almost 10-fold) after overnight exposure. In conjunction with our previous results using intermediate exposure periods, this suggests that the factor limiting reporter responses is likely to be a slow rate of uptake and/or metabolism of the fungicide. We confirm that a potentially toxic metabolite of dithiocarbamate fungicides, namely ethylenethiourea (ETU), has minimal toxicity toward C. elegans, even after prolonged exposure at high concentrations. We demonstrate that exposure to Mancozeb (but not ETU) significantly inhibits larval growth in C. elegans, although this parameter is not markedly more sensitive than reporter induction as a toxicological endpoint. Finally, we have used two-dimensional electrophoresis to show that high concentrations of both Maneb and Mancozeb drastically simplify the protein spot profile compared with controls. However, only in the latter case is there evidence of novel proteins being induced. Both fungicides appear toxic to C. elegans, but only Mancozeb induces a strong heat-shock response.

Caenorhabditis elegans as a biomonitor for immunological stress in nematodes.

An experimental system has been developed using Caenorhabditis elegans (Secernentea: Rhabditida), to monitor immunological stress in nematodes. The transgenic C. elegans strain PC72 carries a lacZ reporter gene fused to a C. elegans hsp16-1 gene, which is inducible for beta-galactosidase activity at the heat stress temperature of 26 degrees C. The investigate the possibility of using PC72 to monitor immunological stress, its surface coat was targeted, to mimic immune attack, by raising immune sera against surface coat components selectively removed by the cationic detergent cetyltrimethylammoniunm bromide. Initially, a highly significant induction of beta-galactosidase activity was seen in PC72 incubated in either surface-reactive or naïve rabbit serum. Complement (C3) was detected over the entire surface of adult PC72 and was thought to be responsible for stress-induction with naïve sera. When the immunoglobulin (Ig)G fraction of naïve sera was used in isolation, no stress-induction was seen. In contrast, a two-fold increase in beta-galactosidase activity was seen in the presence of surface-reactive IgG (SR-IgG) which recognised surface components of between 6 and 40 kDa in western blot. The belief that surface reactive IgG could induce a stress response was reinforced by analysis of hsp-16 protein expression. Cationised ferritin was then used to assess whether stress-induction was truly a surface reactive event; binding of cationised ferritin to the nematode surface also resulted in two-fold induction of beta-galactosidase activity. To investigate the downstream biological effects of stress induction, worm growth and fecundity were measured in the presence of IgG preparations. A significant reduction was seen in both worm length and fecundity only when larvae were incubated in surface-reactive IgG, compared to both naïve IgG and K-medium controls. In conclusion, it would appear that C. elegans is a suitable model to monitor the induction of immunological stress at the level of the nematode surface coat. Given the ability of nematode surface antigens to protect the vaccinated host in animal model systems, and the close phylogenetic relationships which exist between C. elegans and nematodes of medical and veterinary importance, it is conceivable that the immunological targets in or on the surface of C. elegans warrant rapid identification.

Effect of single and paired metal inputs in soil on a stress-inducible transgenic nematode.

A toxicity test using a transgenic strain of the free-living soil nematode Caenorhabditis elegans carrying a stress-inducible beta-galactosidase reporter has been adapted for use in soil biomonitoring. High concentrations (250 microg. g(-1)) of cadmium are required to induce the stress response in worms exposed to Lufa 2.2 soil. Even at relatively high concentrations, the response to copper and zinc additions alone is minimal, yet combinations of cadmium and copper in the test soil induce a larger response than with cadmium alone at the same concentration. In contrast, the addition of both zinc and cadmium induces a lower response than cadmium additions alone. Analysis of the interstitial water suggests that there is preferential occupation by copper of sorption sites in the soil, allowing more cadmium to remain in solution. Conversely, cadmium and zinc would appear to interact similarly with the soil constituents, resulting in an increase of both metals in solution with increased additions to the soil. Aquatic tests mimic the results of the soil test, so it is not increased cadmium availability alone that causes an increased stress response when both cadmium and copper are present. The presence of other metals could reduce the amount of cadmium available, which may be one factor in the zinc moderation of the stress response to cadmium. Intracellular mechanisms may also contribute to the copper enhancement of the stress response to cadmium.http://link. springer-ny.com/link/service/journals/00244/bibs/37n4p503.++ +html

The toxicity of dithiocarbamate fungicides to soil nematodes, assessed using a stress-inducible transgenic strain of Caenorhabditis elegans.

The dithiocarbamate fungicides maneb and mancozeb induce a short-term stress response in a transgenic Caenorhabditis elegans strain (PC72) carrying a reporter lacZ gene under the control of a homologous heat shock (hsp16) promoter. This response can be readily monitored as induced beta-galactosidase activity, either by in situ staining or by a quantitative fluorometric enzyme assay. Particularly strong responses are induced by mancozeb (three- to fivefold above controls at 500 microg mL(-1)), causing acute toxicity at concentrations comparable to those recommended for field application (2 mg mL(-1)). Although much of this fungicide is adsorbed by soil, sufficient (ca. 6%) enters the soil water compartment to cause mild stress in the transgenic worm assay. Among possible metabolites from mancozeb breakdown, neither Mn2+ nor ethylenethiourea (ETU) is particularly toxic even at 10% of the optimum mancozeb dosage. Stress responses to a range of other pesticides are also reported, and in several cases it is clear that a nontarget soil species (here, transgenic C. elegans) may be sensitive to low-level contamination.

Use of stress-inducible transgenic nematodes as biomarkers of heavy metal pollution in water samples from an english river system.

Transgenic strains of the nematode Caenorhabditiselegans, which carry stress-inducible lacZ reporter genes, aremeasurably stressed by exposure to heavy metals in aqueous solution. Thisstress response can be quantified, using enzymatic assays for the reportergene-product (Escherichia coli beta-galactosidase), or estimatedapproximately by in situ staining for beta-galactosidase in exposedworms. Stress responses to heavy metals have been demonstrated both inlaboratory tests using Cd2+ or Hg2+, and also in watersamples taken from a metal-polluted river system in southwest England. TheRiver Carnon flows through an area with an ancient mining history,principally for Sn, but also for Cu and other metals; As, Cd, Al, Mn, and Zn,as well as large amounts of Fe, are all present in these ore bodies. Foursites in the Carnon river basin were compared with respect to theirmacroinvertebrate diversity, physical and chemical characteristics (includingthe concentrations of As, Cd, Al, Cu, Mn, Zn, and Fe). Transgenic worms wereexposed to water samples from these four sites, and also to a 0.33%(v/v) dilution of metal-laden minewater from the principal local mine (WhealJane). Transgene expression was induced in all five cases, though markedlyless so for the least polluted of the sites (which also supported a richermacroinvertebrate fauna). Two different transgenic strains were tested inthis study; strain PC72 (using a homologous hsp16 promoter) isslightly more sensitive to most metal-containing water samples than strainCB4027 (using a heterologous Drosophila hsp70 promoter). Bothtransgenic strains and two different assay methods gave essentially similarresults. These findings demonstrate that transgenic nematodes could provide arapid and simple assessment of aquatic pollution, in that the transgeneresponse is inducible by mixtures of dissolved metals at concentrationsactually encountered in metal-polluted watercourses.

Transgenesis in animal systems: a view from within.

The introduction of 'foreign' or altered genes into animals arouses both scientific and public concern. So too does any extension of this practice to human beings at the somatic (tissue) level, let alone interference in the human germ line (genetic material passed on to future generations). The range of possible genetic modifications, both those in progress and those likely in the near future, is so vast as to make a uniform Christian response untenable. As with other technologies, there are both laudable and dubious uses for genetic engineering in animal systems; it is neither an unmixed blessing nor an unmitigated curse. It is argued that Christians should engage with these issues on a case-by-case basis, giving due weight to the possible risks and concomitant suffering for the animals used, but basing our ultimate approval or disapproval on the congruence of means and aims with those evinced by Jesus in his ministry of healing, teaching and reconciliation.

Calcium moderation of cadmium stress explored using a stress-inducible transgenic strain of Caenorhabditis elegans.

In a transgenic strain of Caenorhabditis elegans carrying a stress-inducible lacZ reporter gene, short-term sublethal exposure to heavy metals activates transgene expression. The transgene response to Cd2+ is strongly inhibited by Ca2+ ions; furthermore, Ca2+ reduces the net accumulation of Cd2+ by worms. Both Ca2+ and a variety of calcium uptake inhibitors (nifedipine, La3+, verapamil) depress the dose response of the transgene to Cd2+. Calcium ionophore (A23187) slightly increases transgene activity in control and Cd2+ treated worms, but has a much larger effect in the case of Mn2+, reflecting its much greater affinity for this ion.

A choice between glycogen and delta-crystallin accumulation is made in glial cells and not influenced by overlying neurons.

Chick-embryo neuroretinal cells convert extensively into lens under low-glucose conditions, but this transdifferentiation process is blocked by high-glucose media. We have previously observed an inverse relationship between the levels of glycogen (a marker of normal retinoglial differentiation) and of delta-crystallin (a lens marker) in such cultures. However, most of the glycogen accumulated under high-glucose conditions is apparently localized in those glial (G) cells underlying clusters of neurons (N cells). We here show that glial-enriched cultures (largely depleted of N cells) both accumulate glycogen and fail to transdifferentiate in high-glucose media. Moreover, glycogen localization in groups of glial cells is unaffected by the absence of N cells. Thus the choice between normal and foreign differentiation pathways is made autonomously within the retinoglial-cell population and is not influenced significantly by the presence or absence of N cells.

Expression in non-lens tissues of an enzyme activity related to the 'lens-specific' protein, delta crystallin.

When chick embryo neutral retina (NR) cells are cultured for long periods in vitro, they undergo extensive transdifferentiation into lens and express the lens protein, delta crystallin. We now demonstrate that this process is accompanied by a change in the chromatin conformation of the delta-gene locus from DNAase1-resistant to DNAase1-sensitive in the nuclei of most cells. Transcripts hybridising to a delta probe are also much more prevalent among the in vitro transcription products from lens or transdifferentiated NR culture nuclei, as compared to nuclei from fresh NR tissue. Published evidence indicates that the chick delta 1 crystallin gene encodes the major structural protein of embryonic lens fibres, whereas the closely related delta 2 gene may encode the urea-cycle enzyme argininosuccinate lyase (ASL). Our present data lends further support to this view. Both immunodetectable delta-related protein(s) and ASL activity are present in fresh embryonic NR tissue, as well as in mouse and Rana liver, and in Rana lens. Our polyclonal anti-delta antibody also cross-reacts with a major constituent of commercial bovine ASL, of the same molecular size as chick delta crystallin. Immunoselection studies suggest that the ASL activity in chick embryonic NR is conferred mainly by the delta-related protein band. So-called 'ectopic' expression of delta crystallin in embryonic NR (and other tissues) may thus involve the delta 2/ASL gene, and could reflect some metabolic requirement for ASL activity.

Effects of Pluronic F-68 on 2-deoxyglucose uptake and amino acid incorporation into chick embryonic fibroblasts in vitro.

The effects of the nonionic surfactant Pluronic F-68 on 2-deoxyglucose uptake and amino acid incorporation in vitro into chick embryonic fibroblasts prepared from day 7 embryos have been studied. During early culture growth (3-10 days), Pluronic supplementation at 0.05% (w/v), but not at 0.1%, stimulated 2-deoxyglucose uptake by 20%-30% above control levels. Pluronic also markedly increased amino acid incorporation, maximally at 3 days of culture (200%-300% above control levels with 0.1% and 0.05% Pluronic, respectively), but to a lesser extent thereafter. These effects of Pluronic were seen only when freshly-prepared solutions were used; 'aged' solutions produced no comparable stimulation of 2-deoxy-glucose uptake or amino acid incorporation.

Stimulatory effects of retinal extract and fibroblast growth factor on lentoidogenesis in cultures of chick embryo neuroretinal cells.

A crude extract prepared from embryonic chick retina stimulates growth and particularly transdifferentiation into lens when added as a supplement to neuroretinal (NR) cultures in vitro. This effect is especially marked when using a medium (H) containing 5% horse serum, where growth factors are likely to be limiting. The level of delta-crystallin (lens marker) production in such cultures increases with the concentration of extract. Using extracts from earlier and later stages of retinal development, there is an age-dependent decline in the extent to which transdifferentiation is stimulated. However, such extracts have little effect on the activity of CAT, a neuronal marker enzyme. These effects are most probably mediated by growth factors present in the retinal extract acting upon Müller glial cells or their precursors in the NR cultures. In support of this suggestion, we show that purified fibroblast growth factor (but not epidermal growth factor) exerts similar effects on both culture growth and delta-crystallin accumulation.

pp60c-src expression in transdifferentiating cultures of embryonic chick neural retina cells.

Chick embryo neural retinal cells transdifferentiate extensively into lens cells when cultured in Eagle's MEM containing horse and fetal calf sera (FHMEM). Such cultures express elevated levels of pp60c-src-associated tyrosine kinase activity relative to parallel cultures prevented from transdifferentiating by the addition of supplementary glucose (FHGMEM) or replacement of MEM by medium 199 (F199). Northern blotting and in vitro translation studies suggest that c-src mRNA levels are only slightly higher in late transdifferentiating (FHMEM) cultures as compared to parallel blocked (FHGMEM or F199) cultures. By immunocytochemical staining, we show that pp60c-src protein is largely localized in cell groups undergoing conversion into lens (i.e. expressing delta crystallin) in late FHMEM cultures. Initial studies of pp60c-src in chick lens tissues during development indicate that higher kinase activity is found in the epithelial cells relative to mature lens fibres. Thus pp60c-src may be expressed both during the differentiation of lens cells in vivo and during the transdifferentiation of neural retina cells into lens in vitro.

Glucose metabolism in transdifferentiating and glucose-blocked cultures of chick embryo neuroretinal cells: an inverse relationship between glycogen and delta-crystallin accumulation.

Chick embryo neuroretinal (NR) cells transdifferentiate extensively into lens when cultured for several weeks in low-glucose (FH) medium, but fail to do so when high levels of supplementary glucose (FHG) are present. We show here that most aspects of glucose metabolism are promoted in high-glucose cultures, including lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activities, 2-deoxyglucose uptake, pentose shunt activity and lactate production. Continuous supplementation of high-glucose cultures with low levels of ouabain (FHGO) significantly lowers 2-deoxyglucose uptake, from FHG levels down towards FH levels, especially during the early stages of NR culture. Much later, extensive transdifferentiation into lentoids (with concomitant delta-crystallin accumulation) occurs in these FHGO cultures, which thus resemble FH rather than FHG controls. Another parameter strongly affected by ambient glucose levels is the accumulation of glycogen. Both glycogen itself and glycogen synthetase activity increase steadily in FHG cultures, but decrease slightly under FH conditions. Glycogen accumulation in FHG cultures is largely confined to glial-like cells, particularly those underlying clusters of neurones. Other studies have shown that glial differentiation in vitro is promoted by histotypic interactions with retinal neurones. Thus high glucose may act in concert with neuronal influences to stimulate or stabilize the normal differentiation of retinal glial cells, whose characteristic features in vivo include glycogen synthesis and storage. Furthermore, we show that supplementation of high-glucose cultures with forskolin or dibutyryl cyclic AMP (both of which promote glycogenolysis) results in a slower rate of glycogen accumulation and in enhanced transdifferentiation into lens. In both respects, the forskolin- and dibutyryl cAMP-supplemented FHG cultures are intermediate between FH and FHG controls. Thus the enhancement of normal glial differentiation in NR cultures by high glucose may inhibit or preclude subsequent transdifferentiation into lens.

Heat Shock May Relieve a Posttranscriptional Block on σ Crystallin Synthesis in Cultures of Chick Embryo Neuroretinal Cells: (neural retina/transdifferentiation/posttranscriptional control/δ crystallin/heat shock).

The lens protein δ crystallin is expressed at high levels in lentoids fromed by the transdifferentiation of chick embryo neuroretinal cells in long-term culture under permissive conditions. Previous work has shown that one non-permissive medium (F199) allows transcription of δ crystallin RNA in late neural retinal cultures, but that these δ transcripts remain confined to the nuclei and are neither translated nor indeed detectable in the cytoplasm. We show here that F199 cultures maintained at 43°c throughout transdifferentiate extensively into lentoids, in contrast to similar cultures at 37°c. Following transfer of late (30 day) F199 cultures from 37°c to 43°c, δ crystallin synthesis becomes increasingly prominent over a period of at least 5 days, suggesting that recovery from heat shock (rather than heat shock per se) may be important in this phenomenon. A similar response can be elicited by chemical agents (sodium arsenite, azetidine 2-carboxylic acid) which induce heat-shock (stress) proteins at 37°c. Treatment of late F199 cultures with actinomycin D does not prevent this increase in δ crystallin synthesis, provided the drug is not added until after the initiation of a heat-shock response (6 hr after temperature shift). This suggests that the observed increase in δ synthesis following heat shock involves primarily the export and/or stabilisation of δ mRNAs already present in the nuclei in late F199 cultures. Possible wider implications of this posttranscriptional level of control over δ crystallin synthesis are discussed.

Stress Protein and Crystallin Synthesis During Heat Shock and Transdifferentiation of Embryonic Chick Neural Retina Cells: (chicken/retina/transdifferentiation/heat shock).

Embryonic chick neural retina responds to heat shock by the synthesis of "stress" polypeptides with molecular weights of 85 and 70 kd. Both stress proteins are synthesised from newly-transcribed messenger RNA. Sodium arsenite induces an additional stress protein of MW 25 kd. The heat shock response does not change during culture and subsequent transdifferentiation, and crystallin synthesis is not coinducible with the heat-shock proteins. We have also examined the pattern of protein synthesis at various stages of culture in both monolayer and aggregate systems; although changes in the protein synthetic profine are evident, there is no stress protein induction above basal levels at any time. Whilst mammalian α crystallin (B2 chain) exhibits considerable homology to four small Drosophila heat-shock proteins, no significant antigenic similarity is apparent between δ crystallin and the major avian heat shock proteins. Thus during transdifferentiation, (a) the crystallin proteins do not behave in a manner analogous to stress proteins; moreover (b) crystallin production is not mediated by stress proteins resulting from a culture-induced stress response.

A lamprey lens protein related to avian and reptilian delta crystallin.

Delta crystallin has hitherto been considered specific to reptiles and birds. Evidence presented in this paper suggests that a major lens protein in adult river lampreys (Lampetra fluviatilis) is related to chick delta crystallin. Both are of similar size (Mr 45-50,000), and the lamprey protein cross-reacts with antibodies against chick delta crystallin in immunodiffusion, immunoelectrophoretic and immunoblotting tests. However, the charge properties and V8 proteolytic fragment patterns of the lamprey protein are markedly different from those of avian and reptilian delta crystallins. Preliminary evidence on the occurrence of delta-related DNA sequences in the genomes of all vertebrates examined, is also discussed.

Differential effects of culture media on normal and foreign differentiation pathways followed by chick embryo neuroretinal cells in vitro.

Three different culture media, Ham's F-12, medium 199, and Eagle's minimal essential medium (MEM), were compared with respect to the expression of neuronal (choline acetyl transferase activity: CAT) and glial (hydrocortisone-induced glutamine synthetase activity; GSase) markers of normal differentiation in cultures of 9-day chick embryo neuroretinal cells, and also with respect to the accumulation of a lens marker (delta crystallin) during so-called 'transdifferentiation' in these cultures. MEM allows transient expression of both CAT and GSase activities in early cultures, but also permits extensive delta crystallin accumulation at later stages. F-12 medium gives somewhat higher levels of CAT and GSase activities, the former being noticeably prolonged as compared with parallel MEM cultures; delta crystallin accumulation, however, is largely inhibited in F-12 cultures. By contrast, medium 199 permits only low levels of CAT and GSase activities, perhaps because the neuronal cells are distributed individually over the glial cell sheet in 199 cultures, rather than forming aggregates as in MEM or F-12 cultures. Medium 199 also blocks delta crystallin accumulation. The results of medium changeover between 'transdifferentiation'-permissive (MEM) and non-permissive (199, F-12) conditions suggest: (a) that potential lens precursor cells (whatever their nature) survive in F-12 medium for prolonged periods without extensive expression of the lens phenotype; (b) that such precursor cells become committed to subsequent differentiation as lens cells between 10 and 20 days of culture in permissive MEM medium (as judged by the accumulation of delta crystallin following transfer into F-12); and (c) that medium 199 can block expression of the lens phenotype even in cells already committed (by the above criteria) to lens differentiation, as for instance after 30 days of preculture in MEM.

Expression of Toxin Receptors on Cell Surfaces in Transdifferentiating Cultures of Neural Retina: (chick embryo neural retina/transdifferentiation/neuronal cell surface toxin receptors/δ crystallin/double fluorescent labelling).

It has often been asked which of the cell types found during the early stages of culturing embryonic chick neural retina can undergo transdifferentiation into lens in vitro. Since neuronal cell-surface toxin receptors are maintained in NR cultures for much longer than internal neuronal enzymes (e.g. choline acetyltransferase), and since the transdifferentiation process can be greatly accelerated by preparing reaggregates of neural retina cells after about 10 days of preculture as "monolayers", a direct test of this question became feasible. 7 or 9 day embryonic chick neural retina cells, precultured for 10-12 days as monolayers, were dissociated and reaggregated under continuous gyration. Reaggregates were maintained for 8 days in the presence of either tetanus toxin or FITC-conjugated α-bungarotoxin, to permit surface-bound toxins to become internalised via receptor turnover. The reaggregates were then dissociated, stained with rabbit antitoxin and FITC-conjugated anti-antibody in the case of tetanus toxin-labelled material, and restained with a rat or mouse antibody against chick δ crystallin followed by the appropriate rhodamine-conjugated anti-antibody. Although both FITC/toxin-labelled cells (putative neurones) and rhodamine/δ crystallin-labelled cells (transdifferentiated lens cells) were abundant, no examples of double-labelled cells were observed with 9 day starting material, and only a very few with 7 day starting material. We conclude that the vast majority of differentiated neuronal cells expressing surface receptors for these toxins do not transdifferentiate directly into lens cells.