Beh CT et al. (1985) Worm Breeder's Gazette "an acid phosphatase activity localized to the gut of C elegans."

Beh CT, McGhee JD

[Worm Breeder's Gazette, 1985]

We have previously described a gut-localized esterase as a biochemical marker with which to study the early events in lineage- specific gene expression (Abstracts #14 and 86 in May, 1985, CSH; also Edgar and McGhee, Developmental Biology, in press). We wanted to find a second gut-specific hydrolase with which to compare the esterase expression and an acid phosphatase appears to be a good candidate. Adult worms are turned into extrudates by cutting in the presence of 2 g/ml of levamisole (see Lewis, et. al., Genetics 95: 905 (1980)), fixed in paraformaldehyde and stained with the following mixture: 50 l of 20 mg/ml of 1-naphthyl phosphate, 100 l of 1 M NaOH, 850 l 0.15 M sodium acetate to which is added 100 l freshly diazotized pararosaniline. Final staining pH is close to 5.0. Staining is usually rapid, intense and gut-localized. However, this staining pattern shows two important differences from the esterase pattern. Whereas esterase staining seems to occur throughout the cytoplasm of all gut cells, phosphatase staining appears localized to the edge of the gut lumen in the region of the brush border. (This same pattern is found in worms grown axenically and therefore does not result from clinging E. coli). Moreover, phosphatase staining is not found in the anterior six cells of the gut (i.e., those cells which derive from the anterior daughters of Ea(1/r)a and Ea(1/r)p cells and which show no later nuclear divisions). Since several cells at the posterior end of the gut also do not stain, there seems to be some symmetry in the phosphatase distribution but we re not yet certain how this correlates with their variable nuclear divisions. In any case, the staining pattern indicates that a spatial decision to express or activate the phosphatase is made relatively late in intestinal development. Under the usual staining conditions, the gut phosphatase appears to be the major phosphatase found in crude extracts of unsynchronized populations. Stained isoelectric focusing gels show one major band ( pI about 4.9) along with several minor bands. Unlike the esterase, the phosphatase is stable at its pI and gels can be left to stain overnight, allowing phosphatase activity to be detected in the equivalent of one worm or less. Preliminary steps in purification are consistent with the phosphatase being a membrane protein (i.e., activity is enhanced by Triton X-100) and Triton increases the apparent Molecular Weight on Sephacryl columns. Phosphatase activity in crude extracts is inhibited by tartrate but not by other phosphatase inhibitors such as alloxan, CuSO4, formaldehyde or EDTA. We will probably follow the same scheme worked out for the gut esterase, namely to purify the enzyme as a first step towards gene cloning and to induce isoelectric focusing mutants to locate the phosphatase gene on the genetic map.

Beh CT et al. (1991) Dev Biol "An acid phosphatase as a biochemical marker for intestinal development in the nematode Caenorhabditis elegans."

Chung MA, McGhee JD, Ferrari DC, Beh CT

[Dev Biol, 1991]

We describe an acid phosphatase enzyme (EC 3.1.3.2) that is localized to the intestine of the nematode Caenorhabditis elegans and that should serve as a convenient biochemical marker for gut differentiation. In adult worms, acid phosphatase activity is located along the edge of the gut lumen in the vicinity of the intestinal brush border. All but the anterior six cells of the intestine stain for phosphatase activity; the nonstaining cells all descend from the Ea(l/r)(a/p)a cells. Acid phosphatase activity is low in oocytes and early embryos but increases substantially when embryos reach late morphogenesis stage; this increase corresponds to the appearance of a major band of acid phosphatase activity detectable on isoelectric focusing gels. We designate this band as the product of the pho-1 gene. The pattern of acid phosphatase expression in several embryonic mutants suggests that pho-1 expression in the developing intestine is lineage autonomous. We induced an isoelectric focusing variant in the pho-1 enzyme and used this to map the pho-1 locus about 1.5 map units to the left of center of chromosome II. We purified the pho-1 enzyme to homogeneity (6500-fold purification; 4% recovery of activity); the pho-1 acid phosphatase is a homodimeric glycoprotein with a subunit molecular weight of 55,000 Da. This paper establishes a new experimental system with which to investigate the molecular basis of lineage-specific gene expression during C. elegans development.

Riordan R et al. (2024) Alzheimers Dement "TMEM106B C-terminal fragments aggregate and drive neurodegenerative proteinopathy in transgenic Caenorhabditis elegans."

Riordan R, Liachko NF, Kraemer BC, McMillan PJ, Han M, Kow RL, Saxton A

[Alzheimers Dement, 2024]

INTRODUCTION: Genetic variation in the lysosomal and transmembrane protein 106B (TMEM106B) modifies risk for several neurodegenerative disorders, especially frontotemporal lobar degeneration (FTLD). The C-terminal (CT) domain of TMEM106B occurs as fibrillar protein deposits in the brains of dementia patients. METHODS: To determine the TMEM CT aggregation propensity and neurodegenerative potential, we generated transgenic Caenorhabditis elegans expressing the human TMEM CT fragment aggregating in FTLD cases. RESULTS: Pan-neuronal expression of human TMEM CT in C. elegans causes severe neuronal dysfunction driving neurodegeneration.  Cytosolic aggregation of TMEM CT proteins accompanied by behavioral dysfunction and neurodegeneration. Loss of pgrn-1 did not modify TMEM CT phenotypes suggesting TMEM CT aggregation occurs downstream of PGRN loss of function. The mechanistic drivers of TMEM106B proteinopathy appear distinct from known modifiers of tauopathy. DISCUSSION: Our data demonstrate that TMEM CT aggregation can kill neurons. TMEM106B transgenic C.elegans provide a useful model for characterizing TMEM106B proteinopathy-mediated neurodegeneration in FTLD. HIGHLIGHTS: Pan-neuronal expression of human TMEM106B C-terminal fragments (TMEM CT) in C. elegans neurons drives a suite of disease-related phenotypes useful for modeling the molecular and cellular features of TMEM106B neuropathology. TMEM CT expression results in extensive TMEM aggregation and accumulation of highly detergent insoluble protein species. TMEM CT expression causes moderate to severe neuronal dysfunction dependent on TMEM CT abundance as measured by stereotypical behavioral readouts. TMEM CT expression drives significant neurodegenerative changes. Dendra2 tagged TMEM exhibits similar properties to untagged TMEM allowing ready visualization of the protein. TMEM CT aggregates accumulate adjacent to but not within lysosomes. PGRN loss of function does not impact TMEM CT toxicity. Modifiers of tau and TDP-43 proteinopathies have little impact on TMEM CT-related neurodegenerative phenotypes.

Ropiak HM et al. (2016) J Agric Food Chem "Structure-activity relationship of condensed tannins and synergism with trans-cinnamaldehyde against Caenorhabditis elegans."

Thamsborg SM, Ramsay A, Williams AR, Desrues O, Ropiak HM, Mueller-Harvey I

[J Agric Food Chem, 2016]

Parasitic gastrointestinal nematodes (GIN) of livestock are increasingly developing resistance to synthetic nematocidal drugs. Moreover, the use of nematocides can induce ecotoxicity by affecting free-living nematodes. Condensed tannins (CT) are a structurally diverse group of bioactive plant compounds possessing anthelmintic activity against GIN. We investigated the relationship between the chemical structure of contrasting, purified CT and nematocidal effects using Caenorhabditis elegans. We also explored whether the nematocidal activity of CT could synergize with trans-cinnamaldehyde (CIN). A non-significant correlation was evident between the ability of CT fractions to inhibit C. elegans motility and the molar proportion of prodelphinidin subunits in purified CT samples. Synergistic inhibition of motility was achieved by combinations of CT and CIN. Galloylation of procyanidins was also a key factor for synergy. To increase the nematocidal effect of CT, plant sources containing CT with specific structural features could be selected and combined with compounds acting in synergy.

Hawkins NC et al. (2003) Biotechniques "Creating precise GFP fusions in plasmids using yeast homologous recombination."

Hawkins NC, Garriga G, Beh CT

[Biotechniques, 2003]

Using a combination of primer amplification, homologous recombination, and yeast genetics, we established a method for creating precise promoter and protein fusions in genes originating from organisms other than yeast. One major advantage of this new method is its versatility. Fusions can be produced within a target gene without constraints regarding the site of insertion. Thus, fusions can be generated within a target sequence exactly at the site desired, and all sequences upstream and downstream of the insertion site were preserved. To illustrate the general applicability of this technique, we fused the gene encoding GFP to a Caenorhabditis elegans homologue of the dishevelled gene, dsh-2. This approach is not restricted to GFP fusions but can be utilized to create fusions between almost any two sequences regardless of the source. Therefore, this method provides a flexible alternative to other PCR-mediated techniques.

Ferrari DC et al. (1987) International C. elegans Meeting "a gut-specific acid phosphatase in C elegans -- expression, biochemistry, and genetics."

Beh CT, Ferrari DC, McGhee JD

[International C. elegans Meeting, 1987]

Li H et al. (2022) Sci Total Environ "Direct toxicity of environmentally persistent free radicals to nematode Caenorhabditis elegans after excluding the concomitant chemicals."

Xing B, Lang D, Zuo N, Pan B, Liu Y, Li H, Steinberg C

[Sci Total Environ, 2022]

Environmentally persistent free radicals (EPFRs) have attracted extensive attention due to their potential toxicity. However, EPFRs-containing particles always coexist with their parent organic contaminants and intermediate degradation products (IM), which may have hindered the toxicity assessment of EPFRs. In this study, the toxicity of EFFRs was specifically verified after comparing the systems without EPFRs, such as the immediate mixture of catechol (CT) and particles, solutions of CT only, IM extracted from the particles, as well as particles after EPFRs quenching. Caenorhabditis elegans (C. elegans) were used as model organisms. Our results showed that EPFRs-containing particles (Si-Al-CT) exhibited significant toxicity to C. elegans, but not for the parent chemical CT and IM on the particles. Higher levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in the Si-Al-CT system were attributed to the mediated generation of -O₂^- and -OH via EPFRs. EPFRs could increase gene expressions related not only to oxidative stress and biotransformation in C. elegans, but also to indications of disturbances in energy homeostasis, survival, proliferation, cell and embryonic development. Overall, these results confirmed the direct toxicity of EPFRs and highlighted the key role of EPFRs which may be neglected in assessing the environmental risks of organic contaminants.

Bjork P et al. (2002) Mol Biol Cell "A novel conserved RNA-binding domain protein, RBD-1, is essential for ribosome biogenesis."

Burglin TR, Bjork P, Wieslander L, Bauren G, Tong YG, Jin S, Hellman U

[Mol Biol Cell, 2002]

Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20-30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

Sahu, Surasri N et al. (2009) International Worm Meeting "A High Throughput Approach Towards Vibrio cholerae Pathogenesis in Caenorhabditis elegans."

Sahu, Surasri N, Cinar, Hediye N

[International Worm Meeting, 2009]

Vibrio cholerae (VC), the causative agent of cholera in humans, is responsible for devastating epidemics and pandemics across the world. The major virulence factor underlying the pathogenesis of cholera is cholera toxin (CT). However, CT negative VC non-O1 and non-O139 strains and CT deleted vaccine mutant strains are still capable of causing disease symptoms through mechanisms that are currently unclear. Vibrio cholerae cause lethality, growth retardation and escape behavior in Caenorhabditis elegans via cholera toxin (CT), and toxin co-regulated pili (TCP) independent process (1, 2). Absence of the CT and TCP response in C. elegans model may help to reveal the role of other toxins of VC that might otherwise be masked by these major virulence factors. CVD110, a V.cholerae vaccine strain, lacking several virulence factors such as zonula occludens toxin (zot), accessory toxin (ace), hemolysin (hly A) and cholera toxin A subunit gene (ctx A), showed attenuated killing in C. elegans (3,4). We are conducting microarray experiments to define host immune response genes expressed upon exposure to VC virulence factors. Differential expression profiling of C. elegans exposed to wild type VC versus CVD110 are being done using Affymetrix expression microarrays. Results of these experiments will be presented. (1) Vaitkevicius K. et al. PNAS, 103 (2006) 9280-9285 (2) Cinar HN. et al. 16 th International C. elegans Meeting, 2007 (3) Michalski J. et al. Infection and Immunity 61 (1993) 4462-4468 (4) Cinar HN. et al. Aging Stress and Pathogenesis Meeting, 2008.

Katiki LM et al. (2013) Vet Parasitol "Anthelmintic effect of plant extracts containing condensed and hydrolyzable tannins on Caenorhabditis elegans, and their antioxidant capacity."

Amarante AF, Gonzalez JM, Zajac AM, Lindsay DS, Katiki LM, Chagas AC, Ferreira JF

[Vet Parasitol, 2013]

Although tannin-rich forages are known to increase protein uptake and to reduce gastrointestinal nematode infections in grazing ruminants, most published research involves forages with condensed tannins (CT), while published literature lacks information on the anthelmintic capacity, nutritional benefits, and antioxidant capacity of alternative forages containing hydrolyzable tannins (HT). We evaluated the anthelmintic activity and the antioxidant capacity of plant extracts containing either mostly CT, mostly HT, or both CT and HT. Extracts were prepared with 70% acetone, lyophilized, redissolved to doses ranging from 1.0mg/mL to 25mg/mL, and tested against adult Caenorhabditis elegans as a test model. The extract concentrations that killed 50% (LC(50)) or 90% (LC(90)) of the nematodes in 24h were determined and compared to the veterinary anthelmintic levamisole (8 mg/mL). Extracts were quantified for CT by the acid butanol assay, for HT (based on gallic acid and ellagic acid) by high-performance liquid chromatography (HPLC) and total phenolics, and for their antioxidant activity by the oxygen radical absorbance capacity (ORAC) assay. Extracts with mostly CT were Lespedeza cuneata, Salix X sepulcralis, and Robinia pseudoacacia. Extracts rich in HT were Acer rubrum, Rosa multiflora, and Quercus alba, while Rhus typhina had both HT and CT. The extracts with the lowest LC(50) and LC(90) concentrations, respectively, in the C. elegans assay were Q. alba (0.75 and 1.06 mg/mL), R. typhina collected in 2007 (0.65 and 2.74 mg/mL), A. rubrum (1.03 and 5.54 mg/mL), and R. multiflora (2.14 and 8.70 mg/mL). At the doses of 20 and 25mg/mL, HT-rich, or both CT- and HT-rich, extracts were significantly more lethal to adult C. elegans than extracts containing only CT. All extracts were high in antioxidant capacity, with ORAC values ranging from 1800 moles to 4651 moles of trolox equivalents/g, but ORAC did not correlate with anthelmintic activity. The total phenolics test had a positive and highly significant (r=0.826, p 0.01) correlation with total hydrolyzable tannins. Plants used in this research are naturalized to the Appalachian edaphoclimatic conditions, but occur in temperate climate areas worldwide. They represent a rich, renewable, and unexplored source of tannins and antioxidants for grazing ruminants, whereas conventional CT-rich forages, such as L. cuneata, may be hard to establish and adapt to areas with temperate climate. Due to their high in vitro anthelmintic activity, antioxidant capacity, and their adaptability to non-arable lands, Q. alba, R. typhina, A. rubrum, and R. multiflora have a high potential to improve the health of grazing animals and must have their anthelmintic effects confirmed in vivo in both sheep and goats.