D. I. De Pomerai

STRESS-INDUCIBLE TRANSGENIC NEMATODES AS BIOMONITORS OF SOIL AND WATER POLLUTION

This paper reviews the current status of nematodes with stress-inducible transgenes as biosensors responsive to a range of external stressors, e.g., soil or water pollution, microwave radiation or immunological attack. TransgenicCaenorhabditis elegans carrying reporter genes under heat shock promoter control express reporter products only under stressful conditions. Although relatively insensitive to single metal ions, these worms respond to complex mixtures present in metal-contaminated watercourses and to laboratory mixtures containing similar constituents, but not to any of their components singly at comparable concentrations. Responses to metal mixtures are enhanced by a non-ionic surfactant, Pluronic F-127. Metals taken up by food bacteria and insoluble metal carbonates can also evoke stress responses, both in soil and aqueous media. However, high concentrations of added metals are needed to induce clear-cut responses in soil, owing to metal sorption onto clays and organic matter. Transgenic worms are also stressed by exposure to microwave radiation; pulsed signals generate responses that diminish markedly with distance from the source. Finally, stress responses are inducible by anti-epicuticle antisera and complement, suggesting that immune attack can also activite the heat shock system. The development of rapid microplate toxicity assays based on transgenic nematodes is discussed.

THE INFLUENCE OF GROWTH‐INHIBITING AND GROWTH‐PROMOTING MEDIUM CONDITIONS ON CRYSTALLIN ACCUMULATION IN TRANSDIFFERENTIATING CULTURES OF EMBRYONIC CHICK NEURAL RETINA

The effects of media containing undialysed serum (controls) or dialysed serum with or without ascorbic acid, were compared during the second half of the 41‐day culture period in embryonic chick neural retina cultures, which had all been grown in control medium prior to 19 days. Conditions permitting greatest culture growth (controls) showed earlier and more extensive development of lentoids, greater accumulation of total crystallin and a higher proportion of δ relative to α+β crystallins. Conditions allowing least culture growth (dialysed serum) gave converse results throughout. Thus changes in culture growth rate apparently affect δ crystallin production more than α or β crystallin production. Insulin promotes growth in neural retina cultures, whether present throughout the culture period (in this case 31 days), or only from 18 days onwards. The frequency and survival of putative neuronal cell aggregates are both increased by insulin during the first 18 days of culture. Delta crystallin production during subsequent transdifferentiation is selectively promoted by insulin when present throughout, but this effect is largely obviated when insulin is present only from 18 days onwards. This anomaly could arise through percursor cell selection during the earlier phases of culture, since it is possible that some (not all) lentoids may be derived from aggregates of neuronal‐like cells in neural retina cultures. Thus precursor cell selection as well as culture growth rate may influence the pattern of crystallin production during transdifferentiation.

Inter and intra-specific genomic variability of the potato cyst nematodes Globodera pallida and G. rostochiensis from Europe and South America using RAPD-PCR

Eleven populations of the potato cyst nematode Globodera pallida from the UK and Peru were analyzed and compared using RAPD-PCR with four 10-mer primers. In addition, seven populations of G. rostochiensis from the UK and single populations from Germany, the Netherlands and Bolivia were included for comparison. Genomic variability was found both between and within species, with more variability in G. pallida than G. rostochiensis. The genomic pool of British G. pallida populations showed considerably less variation than the Peruvian populations, with 73% and 41% similarity between populations respectively. The genomic similarity among populations of G. rostochiensis was 89% for UK populations and 82% when the two continental European populations were included. Nevertheless, between populations within each species and from the same locality, genomic differences were still found.

Determination of Chick Neuro-retinal Cells in Culture: Serum Factors Acting between 12 and 20 Days of Culture Influence the Extent of Subsequent Lens Cell Formation

Embryonic chick neuroretinal cells transdifferentiate into lens cells during culture in media containing foetal calf serum (F). This process is largely inhibited if horse serum plus supplementary glucose (Hg) is substituted for F. This paper explores the effect of medium changeover (from F to Hg or vice versa) on the subsequent appearance of lens‐specific δ‐crystallin. If cultures are changed from Hg to F up to 12 days of culture, δ‐production at 40 days is similar to that for controls maintained in F throughout. Changeovers between 14 and 17 days progressively inhibit subsequent δ production, and after 19 days in Hg, lens transdifferentiation cannot be induced by F. Conversely, if cultures are maintained in F for up to 17 days, a changeover to Hg blocks transdifferentiation, whereas similar transfers performed after 19 days give increased δ production. These results suggest that some retinal cells which will eventually form lens in vitro become so determined between the 12th and 20th days of culture. A mixture of 50% Hg and 50% F medium (FHg) does not support δ production even after 60 days, but in the absence of supplementary glucose (FH), δ appears in considerable amounts by 30 days.

A switch for transdifferentiation in culture: Effects of glucose on cell determination in chick embryo neuroretinal cultures☆

Abstract Chick embryo neuroretinal cells accumulate lens-specific δ-crystallin when cultured in Eagles minimal essential medium containing 5% foetal calf serum and 5% horse serum (FH), but fail to do so if supplementary glucose is present (FHG). Culture growth rates are similar in the two media, but choline acetyltransferase activity is maintained for longer in FHG medium. By transferring cultures from FHG into FH medium and vice versa, we show that potential lens precursor cells survive for at least 21 days in FHG medium, while some retinal cells become irreversibly committed to crystallin production after 18 days in FH medium.

Developing a rapid throughput screen for detection of nematicidal activity of plant cysteine proteinases: the role of Caenorhabditis elegans cystatins

Plant cysteine proteinases (CPs) from papaya (Carica papaya) are capable of killing parasitic nematode worms in vitro and have been shown to possess anthelmintic effects in vivo. The acute damage reported in gastrointestinal parasites has not been found in free-living nematodes such as Caenorhabditis elegans nor among the free-living stages of parasitic nematodes. This apparent difference in susceptibility might be the result of active production of cysteine proteinase inhibitors (such as cystatins) by the free-living stages or species. To test this possibility, a supernatant extract of refined papaya latex (PLS) with known active enzyme content was used. The effect on wild-type (Bristol N2) and cystatin null mutant (cpi-1(-/-) and cpi-2(-/-)) C. elegans was concentration-, temperature- and time-dependent. Cysteine proteinases digested the worm cuticle leading to release of internal structures and consequent death. Both cystatin null mutant strains were highly susceptible to PLS attack irrespective of the temperature and concentration of exposure, whereas wild-type N2 worms were generally resistant but far more susceptible to attack at low temperatures. PLS was able to induce elevated cpi-1 and cpi-2 cystatin expression. We conclude that wild-type C. elegans deploy cystatins CPI-1 and CPI-2 to resist CP attack. The results suggest that the cpi-1 or cpi-2 null mutants (or a double mutant combination of the two) could provide a cheap and effective rapid throughput C. elegans-based assay for screening plant CP extracts for anthelmintic activity.

The effects of organic pesticides on inner membrane permeability in Escherichia coli ML35

We have tested whether some pesticides might cause inner membrane leakage in ML35 Escherichia coli cells, which express β-galactosidase (lacZ; EC 3.2.1.23) constitutively but lack the permease (lacY) required for substrate entry. The activity of β-galactosidase (indicative of substrate leakage through the inner membrane) was increased by various concentrations of pesticides, including the organometallic fungicides maneb and mancozeb, the insecticide Thiodan, and the herbicide Ally, as well as by antibiotics such as ampicillin, gramicidin D, and the calcium ionophore A23187. The enzyme activity was increased by up to ∼30% when the E. coli ML35 strain was exposed to various concentrations (between 50 and 250 ppm) of both fungicides. Thiodan had only a slight effect on β-galactosidase activity (increase of 12.8%), whereas, among the antibiotics, the calcium ionophore at 20 μg/ml caused a significant increase in enzyme activity by up to 61.8%. This effect is similar to that of sodium dodecyl sulfate, used as positive control (∼70% increase). Accumulation of maneb and mancozeb by bacterial cells was also studied taking advantage of their metal content and using atomic absorption spectrophotometry. In parallel with the increase in enzyme activity, both fungicides accumulated in the cells as a function of their concentration. Time course experiments (3, 6, and 9 h) of fungicide accumulation and of bacterial growth at various pesticide concentrations were also carried out. Maneb seems to inhibit the bacterial growth better than mancozeb. In addition, maneb uptake increases with time up to 9 h at all tested concentrations, whereas the accumulation of mancozeb is similar at all the exposure times tested. This indicates a different uptake and/or metabolizing strategy by E. coli cells for the two fungicides.

The measurement of immunological stress in nematodes

No article involving the nematode cuticle can ignore the work of Donald Lee. Since the publication of his book Physiology of nematodes (Lee, 1965), he has provided us with an extensive knowledge of the structure, composition, maintenance and growth of a variety of nematode cuticles and has undoubtedly contributed a great deal to our understanding of this unique and diverse structure.

Heat Shock May Relieve a Posttranscriptional Block on σ Crystallin Synthesis in Cultures of Chick Embryo Neuroretinal Cells

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.

Two-step control of δ-crystallin gene expression during in vitro transdifferentiation of chick embryo neural retina cells

Abstract Chick embryo neural retina cells cultured in Eagles MEM (FHMEM) transdifferentiate extensively into lens cells, synthesising high levels of the characteristic lens protein, δ-crystallin. However, such cultures fail to transdifferentiate when maintained in FHMEM plus supplementary glucose (FHGMEM), or when MEM is replaced by medium 199 (F199). Using [35S]methionine in vitro labelling of cultures and immunoblotting, we have monitored δ-crystallin synthesis and accumulation in the three types of medium. In addition, we have used in vitro translation and a δ-crystallin-cDNA clone hybridised both to Northern blots and to cultures in situ, in order to investigate the level of δ-crystallin transcripts in cellular RNA populations. In FHMEM cultures, transdifferentiation is preceded by a brief phase of δ transcript accumulation within the nuclei occurring at around Day 20 of culture, after which transcripts are also found in the cytoplasm. In FHGMEM cultures, δ transcripts do not accumulate to significant levels at any stage of culture. By contrast, in F199 cultures δ transcripts are accumulated in a subpopulation of cells, but they remain largely confined to the nuclei even in late stage cultures. Thus these nonpermissive media (FHGMEM and F199) indicate two different levels at which δ-crystallin gene expression can be controlled during transdifferentiation.