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[
Worm Breeder's Gazette,
1993]
We have developed a new procedure for recovering nematodes from soils in an efficient and non-destructive manner, which has permitted the development of a short-term soil toxicity test using nematodes as toxicity indicators. The procedure involves gentle centrifugation through a colloidal silica suspension (Ludox AM or Ludox HS-40, both available from E. I. Du Pont de Nemours & Co., Wilmington, DE), which allows the soil to settle out while floating the nematodes on top of the suspension. The nematodes are recovered with great efficiency (~90%) and are unharmed by the recovery process. Short-term lethality tests (24-hour exposure) were performed with several metals (Cd, Cu, Hg, Ni, Pb, and Zn) in several different characterized soils, and replicable results were obtained, allowing concentration-response curves to be generated and LC(50)s to be estimated. It was found that the presence of soil generally decreased the toxicity of the metals to nematodes when compared to tests done without soil present. This is presumably due to the sorption of the metal ions to the soil particles, which lowered their availability to the nematodes. Comparison between nematode lethality results and data published for earthworms exposed to metals in soil revealed that, in most cases, the nematode is at least as sensitive as the earthworm in terms of its response to toxic metals in soil. The earthworm test, which is currently the standard animal soil toxicity test, has the disadvantage of taking two weeks to perform, while the nematode test can be done in 24 hours. Some correlations between soil or metal properties and the resulting lethal effects were obtained, while other properties showed no significant correlation with toxic effects. This suggests that, as other studies have indicated, the bioavailability of metals in soils cannot be reliably predicted by considering only a few sample conditions. Soils are complex and heterogeneous systems, and there are many physico-chemical processes that may occur within them and which may influence the resulting bioavailability of a contaminant. The C. elegans soil toxicity test may provide the means to identify some of the more important environmental characteristics that influence contaminant bioavailability.
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[
Worm Breeder's Gazette,
1988]
In a screen for monoclonals with specificity for embryonic antigens, H. B. and E. H. fortuitously found two hybridoma lines that produce antibodies that bind specifically to the L1 cuticle in squashes of mixed stages. S. D. and S. P. have partially characterized the binding to live worms and antigens in extracts of L1 cuticles. Antibodies were elicited by in vitro immunization of a primary mouse splenocyte culture with a mixture of embryonic antigens. Hybridomas were screened by indirect immunofluorescence of Bristol strain worms in squashes fixed on microscope slides (Albertson, Sulston, and Hedgecock, WBG Vol. 7, #1, p. 73). Two hybridomas were found that stained L1 cuticles specifically. Only small larvae with L1 type alae stained; other structures or stages never stained. After cloning by limiting dilution and rescreening, these cell lines were grown in quantity and immunoglobulins isolated from culture supernatants by 0- 50% saturated ammonium sulfate precipitation. Resuspended and dialyzed ammonium sulfate fractions are the standard antibody solutions used in the experiments described here. Immunoglobulin class was determined by immunodiffusion (Ouchterlony). Both antibodies (M37 and M38) precipitated specifically with sheep antimouse IgM serum. In subsequent indirect antibody binding experiments, goat anti-mouse IgM secondary antibodies were used. Binding of the antibodies to live L1's in immunofluorescence tests is strongly temperature-dependent. When antibody incubations with live animals are done at room temperature, little binding is observed. If antibody incubations are done at 4 C or at 0 C, binding is quite uniform initially, but the antibody stain 'flakes off' in big flakes like paint flaking off a wall as the slide warms up. After flaking has occurred, the same sample can be washed and restained, indicating that some antigen is still present on the surface. We do not know whether the flaking involves dissociation of the antibody without antigen removal, or whether antigen comes off too. However, heat- killed L1's can also be restained after flaking, indicating that restaining does not require active replacement of the antigen by living worms. Antibody binding to worms fixed in squashes exhibited no similar temperature dependence. L1's were obtained in large numbers (up to 10+E6) for isolation of cuticle proteins. Eggs obtained by Cloroxing were hatched overnight in M9. L1's were harvested and cuticles and cuticle proteins were isolated after sonication by standard procedures. Sonication supernatant, SDS extract, and SDS- ME extract were tested for antibody binding in a 'dot blot' assay. Cuticle extracts were dotted onto nitrocellulose and incubated sequentially with M37 or M38, alkaline phosphatase- or horseradish peroxidase-conjugated goat anti-mouse IgM, and enzyme substrate. Antigen was detected predominantly in the SDS extract of L1 cuticles. Antigen was not detected in similar extracts of adult cuticles. Antibody binding to the SDS extract showed a distinct optimum of pH 5-6 for the primary antibody incubation. Cuticle extracts were separated on 12% Laemmli SDS-PAGE slab gels and electrotransferred to nitrocellulose. Gels were incubated with antibody using conditions optimized in the dot blot. As before, antibody binding was observed only in an SDS extract of L1 cuticles. The predominant antigenic species formed a 'ladder' of about ten sharp, equally spaced bands centered around 20,000 daltons in MW. Less reproducibly, higher molecular weight antigenic bands appear. Appearance of the lower molecular weight ladder is insensitive to predigestion of the sample with protease K. Currently, our working hypothesis is that the antigen recognized by M37-M38 (both produce similar patterns in the Western blot experiment) is a stable, non- protein moiety that may be attached to a labile cuticle surface protein molecule.
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[
J Nematol,
1995]
An aquatic 24-hour lethality test using Caenorhabditis elegans was used to assess toxicity of glucosinolates and their enzymatic breakdown products. In the absence of the enzyme thioglucosidase (myrosinase), allyl glucosinolate (sinigrin) was found to be nontoxic at all concentrations tested, while a freeze-dried, dialyzed water extract of Crambe abyssinica containing 26% 2-hydroxyl 3-butenyl glucosinolate (epi-progoitrin) had a 50% lethal concentration (LC) of 18.5 g/liter. Addition of the enzyme increased the toxicity (LC value) of sinigrin to 0.5 g/liter, but the enzyme had no effect on the toxicity of the C. abyssinica extract. Allyl isothiocyanate and allyl cyanide, two possible breakdown products of sinigrin, had an LC value of 0.04 g/liter and approximately 3 g/liter, respectively. Liquid chromatographic studies showed that a portion of the sinigrin decomposed into allyl isothiocyanate. The results indicated that allyl isothiocyanate is nearly three orders of magnitude more toxic to C. elegans than the corresponding glncosinolate, suggesting isothiocyanate formation would improve nematode control from application of glucosinolates.
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[
International C. elegans Meeting,
1989]
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[
Archives of Environmental Contamination and Toxicology,
1993]
A new method for recovering nematodes from soils in an efficient, reproducible, and non-destructive manner has been developed. It was used to conduct short-term soil toxicity tests using the soil-dwelling nematode Caenorhabditis elegans and several different soil types spiked with copper chloride. The recovery method, which involves centrifugation through a colloidal silica suspension, allows the nematodes to be extracted from the soil matrix so that lethality can be assessed. The nematodes are unharmed by the recovery procedure, and both live and dead individuals are recovered with high efficiency (well over 80%), allowing reproducible concentration-response curves to be made after a 24-h exposure. The LC50s for copper were increased about tenfold by the presence of soil, and different soils had significantly different effects on toxicity. Toxicity of copper ion was also influenced by the concentration of sodium chloride and potassium chloride in the test solution, and the presence of bacteria increased the toxicity of copper ion in some soils. The LC50s in soil were close to the LC50 for the 2-week earthworm soil toxicity test, suggesting that a 24-h nematode toxicity test may be comparable to the 2-week earthworm test in
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[
Environmental Toxicology and Chemistry,
1995]
This study used a randomized block design to investigate the importance of several variables in using the free-living soil nematode Caenorhabditis elegans for aquatic toxicity testing. Concentration-response data were obtained on nematodes of various developmental stages exposed to four metals (Cd, Pb, Cu, and Hg) and a water-soluble organic toxicant, sodium pentachlorophenate (PCP), under conditions of varied solvent medium (with or without salts and with or without a bacterial food source). The end points measured were 24- and 96-h mortality LC50 value, as well as development of larval stages to adulthood and evidence of reproduction. The results suggest that nematodes of various ages respond similarly to a given toxicant for all end points measured, although adults cultured from eggs appeared more sensitive than adults cultured from dauer larvae. The most important environmental variable in determining toxicity was the medium in which the tests were conducted. The presence of potassium and sodium salts in the medium significantly (p < 0.05) reduced the toxicity of many test samples. The presence of bacteria had little effect on 24-h tests with salts, but was important in 96-h survival and development. Based on sensitivity and ease of handling, adults cultured from eggs are recommended in both 24-h and 96-h tests.
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[
Water Air and Soil Pollution,
1994]
A previously developed soil toxicity test for rapidly determining the toxicity of chemicals to the soil-dwelling nematode Caenorhabditis elegans was used to measure the toxicity of four metals (Zn 2+, Cd 2+, Cu 2+, and Pb 2+) added to four soils common to the southeastern United States. Nematode survival after a 24-hour exposure in the presence of a bacterial food source was assessed. All soils reduced the toxicity of most metal ions compared to solutions without soil. Pb was the most strongly affected, while Cd toxicity was not much influenced by the soils. Correlations between the LC50s and various soil or metal characteristics were determined. No significant correlation was found between LC50s and many soil characteristics commonly cited as having large effects on soil bioavailability of metals. Although sample size was limited, the indication was that bioavailability of metals to nematodes is determined by a complex array of many interacting soil, as well as metal, properties. Comparision of the relative mobilities of these ions in other soils with the relative toxicity measured here suggests that mobility may be a good predictor of toxicity. The C. elegans soil toxicity test is shown to be as sensitive and more rapid than the commonly used earthworm
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[
J Ginseng Res,
2017]
BACKGROUND: Heat-processed ginseng, sun ginseng (SG), has been reported to have improved therapeutic properties compared with raw forms, such as increased antidiabetic, anti-inflammatory, and antihyperglycemic effects. The aim of this study was to investigate the antiobesity effects of SG through the suppression of cell differentiation and proliferation of mouse 3T3-L1 preadipocyte cells and the lipid accumulation in Caenorhabditis elegans. METHODS: To investigate the effect of SG on adipocyte differentiation, levels of stained intracellular lipid droplets were quantified by measuring the oil red O signal in the lipid extracts of cells on differentiation Day 7. To study the effect of SG on fat accumulation in C.elegans, L4 stage worms were cultured on an Escherichia coli OP50 diet supplemented with 10g/mL of SG, followed by Nile red staining. To determine the effect of SG on gene expression of lipid and glucose metabolism-regulation molecules, messenger RNA (mRNA) levels of genes were analyzed by real-time reverse transcription-polymerase chain reaction analysis. In addition, the phosphorylation of Akt was examined by Western blotting. RESULTS: SG suppressed the differentiation of 3T3-L1 cells stimulated by a mixture of 3-isobutyl-1-methylxanthine, dexamethasone, and insulin (MDI), and inhibited the proliferation of adipocytes during differentiation. Treatment of C.elegans with SG showed reductions in lipid accumulation by Nile red staining, thus directly demonstrating an antiobesity effect for SG. Furthermore, SG treatment downregulated mRNA and protein expression levels of peroxisome proliferator-activated receptor subtype (PPAR) and CCAAT/enhancer-binding protein-alpha (C/EBP) and decreased the mRNA level of sterol regulatory element-binding protein 1c in MDI-treated adipocytes in a dose-dependent manner. In differentiated 3T3-L1 cells, mRNA expression levels of lipid metabolism-regulating factors, such as amplifying mouse fatty acid-binding protein 2, leptin, lipoprotein lipase, fatty acid transporter protein 1, fatty acid synthase, and 3-hydroxy-3-methylglutaryl coenzyme A reductase, were increased, whereas that of the lipolytic enzyme carnitine palmitoyltransferase-1 was decreased. Our data demonstrate that SG inversely regulated the expression of these genes in differentiated adipocytes. SG induced increases in the mRNA expression of glycolytic enzymes such as glucokinase and pyruvate kinase, and a decrease in the mRNA level of the glycogenic enzyme phosphoenol pyruvate carboxylase. In addition, mRNA levels of the glucose transporters GLUT1, GLUT4, and insulin receptor substrate-1 were elevated by MDI stimulation, whereas SG dose-dependently inhibited the expression of these genes in differentiated adipocytes. SG also inhibited the phosphorylation of Akt (Ser473) at an early phase of MDI stimulation. Intracellular nitric oxide (NO) production and endothelial nitric oxide synthase mRNA levels were markedly decreased by MDI stimulation and recovered by SG treatment of adipocytes. CONCLUSION: Our results suggest that SG effectively inhibits adipocyte proliferation and differentiation through the downregulation of PPAR and C/EBP, by suppressing Akt (Ser473) phosphorylation and enhancing NO production. These results provide strong evidence to support the development of SG for antiobesity treatment.
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[
Anal Chem,
2021]
Because of the lack of facile and accurate methods to track stress granule (SG) dynamics in live cells and <i>in vivo</i>, in-depth studies of the biological roles of this attractive membraneless organelle have been limited. Herein, we report the first small-molecule probe, <b>TASG</b>, for the selective, convenient and real-time monitoring of SGs. This novel molecule can simultaneously bind to SG RNAs, the core SG protein G3BP1, and their complexes, triggering a significant enhancement in fluorescence intensity, making <b>TASG</b> broadly applicable to SG imaging under various stress conditions in fixed and live cells, <i>ex vivo</i> and <i>in vivo</i>. Using <b>TASG</b>, the complicated endogenous SG dynamics were revealed in both live cells and <i>C. elegans</i>. Collectively, our work provides an ideal probe that has thus far been absent in the field of SG investigations. We anticipate that this powerful tool may create exciting opportunities to investigate the underlying roles of SGs in different organisms.
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[
International Worm Meeting,
2021]
GLA-3 is the C. elegans homolog of the mammalian tristetraprolin (TTP) which encodes a protein that contains two CCCH-like zinc-finger domains that function either as transcription factors or RNA-binding proteins. This gene which encodes two splice variants GLA-3A and GLA3B and the protein is expressed in both germline and soma. Loss of
gla-3 function present different alterations: protein degradation in muscle leading to a progressive loss of motility, increased germ cell death by apoptosis, severe defects in meiosis progression, reduced brood size and a low frequency of embryonic lethality. Stress granules (SG) are dynamic cytoplasmic membrane-less organelles that are formed under harsh conditions. SG are composed of mRNAs that are stalled in translation pre-initiation complexes and different types of mRNA binding proteins like TIA-1 and TTP. It has been probed by immunoprecipitation that GLA-3A-B associates with the MAP kinase protein MPK-1/ERK, which is required for pachytene progression during oogenesis, but this association is poorly understood. Additionally, in mammals was observed that TTP is phosphorilated by ERK and this modification affects SG formation, however this interaction has not been studied in C. elegans. Since TTP has been shown to be associated with SG, the aim of this work is to study the function of GLA-3 and to identify whether this protein forms SG under different adverse conditions and the role of MPK-1 / ERK in SG formation. We tested different stress conditions: heat shock (31oC for 3 hours), starvation (6 hours without bacteria) and oxidative stress (1 hour in Paraquat 0.2 mM) and evaluated SG formation using the strain
tn1734 which had the protein GFP fused whit GLA-3A ([gfp::3xflag::
gla-3a]). It was proved that GLA-3 express in germ line from larval stage L2 to adulthood, and in adult gonads, GFP::GLA-3A form SG under heat shock, starvation and oxidative stress. We also observed that iRNA knock-down by feeding animals with dsRNA for
mpk-1 prevent the formation of GLA-3 SG. We conclude that GLA-3 is a SG component and MPK-1 participate in SG assembly.