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[
Worm Breeder's Gazette,
1993]
5-HT is found in a number of neurons in C elegans including the Hermaphrodite Specific Neurons (HSN) which innervate the sex muscles of hermaphrodites and control egg-laying (Desai et al., 1988, Nature 336, 638-646). The addition of 5-HT to worms in M9 results in stimulation of egg-laying and mutants (e.g.. egl-l,
egl-5 and
egl-10 )with HSN abnormalities become bloated with eggs but respond to exogenously applied 5-HT by laying eggs, demonstrating an important regulatory role for 5-HT (Trent et al.,1983, Genetics 104, 619-647). We are interested in the inactivation of biogenic amines in nematodes and have studied the metabolism of 5-HT in C. elegans. Initially, we incubated cut worms with [14C]5-HT and after 2h at 20 C, the radiolabelled metabolites were extracted and analyzed .together with marker compounds on two dimensional silica TLC. Autoradiograms of the developed plates identified the major metabolite as N-acetyl 5-HT and the identity was confirmed by chromatography on two different reversed phase HPLC systems. There were no metabolites corresponding to products of oxidative deamination. N-acetylation of [14C]5-HT by C. elegans homogenate was dependent upon the addition of acetyl CoA to the incubation mixture. The N-acetyltransferase (NAT) was localized in a cytosol fraction and displayed Michaelis-Menten kinetics with a high affinity for 5HT (apparent Km, 30 +/- 8 mM; +/- SEM, n=3). Soluble enzyme was prepared from staged worms (L1, L2 ,L3 ,L4 and adults) and the specific activity of the NAT in each preparation determined from linear reaction rates (fig.). The specific activity was very low (< 8 nmol NA-5HT/h/mg of protein) in L1 and L2 stages but increased in an exponential manner with development, reaching the highest activity ( 144 nmol NA-5HT/h/mg of protein) in adult worms. [See Figure] The increase in the specific activity of the 5-HT NAT occurring at around the L4 /adultstage correlates with the appearance of 5-HT in the HSN cells and the appearance of the sex muscles that are innervated by these neurons. This correlation and the high affinity for 5-HT is consistent with a physiological role for the NAT in the metabolism of this amine but it is also possible that other biogenic amines, such as dopamine and octopamine, can also serve as substrates for this enzyme. We are also considering the possibility that N-acetylated amines are not necessarily end-products of metabolism but may have a signaling role in their own right. Future work will focus on defining the precise function of this enzyme in amine metabolism in adult nematodes.
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[
International C. elegans Meeting,
1995]
We have previously shown that nematodes have high levels of arylalkylamine N-acetyltransferase activity and that N-acetylation could be a primary route for regulating biogenic amine levels in nematodes. Three arylalkylamine N-acetyltransferase enzymes have recently been identified and purified to electrophoretic homogeneity from a whole worm homogenate preparation of Ascaridia galli using ammonium sulphate precipitation followed by hydrophobic interaction, metal chelate and ion exchange chromatography. A major peak of activity was associated with a 40 kDa protein band on SDS-PAGE and we now report the characterisation of this enzyme. The enzyme activity was very labile and showed a strong dependency on the presence of dithiothreitol for activity during purification. The enzyme had ability to N-acetylate arylalkyl-, phenyl-, and catechol-amines and ,furthermore, the N-acetylation of 5-HT was inhibited in a dose-dependent manner by amines from these groups. Enzyme activity was maximal at pH 6.5 when measured at 37 oC with tyramine and acetyl CoA as substrates. The enzyme had a high affinity for tyramine (Km 6.6 *M) and was inhibited by Cu2+, Zn2+ and Hg2+ions (1mM). The Km for acetyl CoA was 18.8 *M. Arylamines and hydrazines, substrates for the mammalian liver acetyltransferase enzymes, did not inhibit or markedly affect the enzyme activity towards tyramine indicating that the A. galli arylalkylamine N-acetyltransferase enzyme (40 kDa) is not similar to the mammalian liver arylamine N-acetyltransferase enzymes. The other two arylalkylamine N-acetyltransferase enzyme activities are being characterised and their properties will also be reported.
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[
Biochem Soc Trans,
1993]
It is generally assumed that biogenic amines function as neurotransmitters and/or hormones in nematodes. Histochemcial techniques have been used to demonstrate the existence of aminergic neurons in a variety of nematodes and a number of studies have reported the presence of biogenic amines in extracts of nematode tissues. There is little known about the precise roles and mechanisms of action of many of these putative neurotransmitters in nematodes, apart from serotonin (5-HT), which appears to be an important intercellular signalling molecule in the free-living nematode, Caenorhabditis elegans and the parasitic nematode, Ascaris suum.
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[
International C. elegans Meeting,
1991]
Oxidative deamination and O-methylation are important reactions in the catabolism of biogenic amines in vertebrates. Although, oxidative deamination is generally considered to be a major pathway for the inactivation of 5-HT and catecholamines in animal parasitic nematodes, this reaction has not been detected in a number of nematode species, including C. eleaans. We have shown, by incubating cut nematodes (C. eleaans, Ascaridia aalli, Brugia pahanqi and Nippostronaylus brasiliensis) with radiolabelled amine, that 5-HT, octopamine and dopamine are taken up by nematode tissues and are metabolised to their N-acetylated derivatives. The N-acetylation reaction requires acetyl-coenzyme A as a co-factor and the activity is localised in a soluble fraction. As yet, we have found no evidence for the involvement of oxidative deamination in the metabolism of these amines in nematodes. No monoamine oxidase activity could be detected in mitochondrial fractions prepared from whole nematodes. Some of the properties of the amine Nacetyltransferase from the parasitic nematode, A. aalli, are described. A single enzyme appears to be responsible for the N-acetylation of 5-HT and octopamine and this activity requires acetyl CoA as a co-factor. The N- acetyltransferase is a soluble enzyme and has an Mr of around 23,000. It has a higher affinity for octopamine (Km, 33uM) compared to 5-HT ( Km, 550uM). The activity towards octopamine is competitively inhibited by tyramine, dopamine and noradrenaline suggesting that these catecholamines may also serve as substrates for this arylalkylamine N- acetyltransferase.
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[
European Worm Meeting,
1998]
The Glutathione S-Transferases (GST) are a superfamily of enzymes that catalyse the nucleophilic addition of the tripeptide glutathione (GSH) to endogenous and xenobiotic electrophilic substrates. GSTs are principally detoxification enzymes, but have been implicated in the development of resistance to a range of pesticides, herbicides and a number of drugs. These enzymes also serve as non-catalytic carrier proteins (ligandins). Recently, a sigma class related GST isolated from Ascaridia galli, displayed a high level of specific activity in the GSH dependent isomerisation of prostaglandin H to prostaglandin E (1). This suggests that certain sigma GSTs may be involved in eicosanoid metabolism. We are investigating the possible functions of members of the sigma class GST of C.elegans. To date, seven sigma class GST-like genes have been identified in C.elegans. We have amplified the corresponding cDNA for each gene by PCR from a cDNA library, and successfully expressed six of the seven cDNA sequences in Escherichia Coli. The recombinant protein product of the R03D7.6 gene has GSH binding properties, allowing purification by affinity chromatography. This protein also shows high levels of activity towards the universal GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). We intend to utilise each recombinant protein firstly to raise monoclonal antibodies to localise the native enzymes in vivo. Secondly, we will conduct a series of assays to analyse the substrate specificities of each recombinant protein. In addition we would like to inject combinations of dsRNAs prepared from the GST cDNAs, to study the effects of interfering with the expression of this GST family. 1 Meyer D.J., Muimo R., Thomas M., Coates D. and Isaac R.E. (1996) Biochem. J. 313, 223-227
-
[
International C. elegans Meeting,
1999]
The Glutathione S-Transferases (GST) are a superfamily of enzymes that catalyse the nucleophilic addition of the tripeptide glutathione (GSH) to endogenous and xenobiotic electrophilic substrates. GSTs are principally detoxification enzymes, but have been implicated in the development of resistance to a range of pesticides, herbicides and a number of drugs. These enzymes also serve as non-catalytic carrier proteins (ligandins). Recently, a sigma class related GST isolated from Ascaridia galli , displayed a high level of specific activity in the GSH dependent isomerisation of prostaglandin H to prostaglandin E 1 . This suggests that certain sigma GSTs may be involved in eicosanoid metabolism. We are investigating the possible functions of members of the sigma ( s ) class GST of C.elegans . 20 s class GST-like genes have been identified in C.elegans . We have amplified the corresponding cDNA for 7 of these genes by PCR from a cDNA library, and successfully expressed six of the seven cDNA sequences in Escherichia coli . The recombinant protein product of the R03D7.6 gene has GSH binding properties, allowing purification by affinity chromatography. This protein also shows high levels of activity towards the universal GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). Antibodies raised to recombinant R03D7.6 have been used to immunoblot GST protein in crude extracts of mixed stage C. elegans and in column purified native GST, demonstrating that R03D7.6 is expressed at detectable levels. The antibody is being used to validate the expression pattern obtained with nematodes transformed with a R03D7.6:: lacZ promoter construct. 1 Meyer D.J., Muimo R., Thomas M., Coates D. and Isaac R.E. (1996) Biochem. J. 313 , 223-227
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[
Zootaxa,
2022]
Rhagovelia medinae sp. nov., of the hambletoni group (angustipes complex), and R. utria sp. nov., of the hirtipes group (robusta complex), are described, illustrated, and compared with similar congeners. Based on the examination of type specimens, six new synonymies are proposed: R. elegans Uhler, 1894 = R. pediformis Padilla-Gil, 2010, syn. nov.; R. cauca Polhemus, 1997 = R. azulita Padilla-Gil, 2009, syn. nov., R. huila Padilla-Gil, 2009, syn. nov., R. oporapa Padilla-Gil, 2009, syn. nov, R. quilichaensis Padilla-Gil, 2011, syn. nov.; and R. gaigei, Drake Hussey, 1947 = R. victoria Padilla-Gil, 2012 syn. nov. The first record from Colombia is presented for R. trailii (White, 1879), and the distributions of the following species are extended in the country: R. cali Polhemus, 1997, R. castanea Gould, 1931, R. cauca Polhemus, 1997, R. gaigei Drake Hussey, 1957, R. elegans Uhler, 1894, R. femoralis Champion, 1898, R. malkini Polhemus, 1997, R. perija Polhemus, 1997, R. sinuata Gould, 1931, R. venezuelana Polhemus, 1997, R. williamsi Gould, 1931, and R. zeteki Drake, 1953.
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[
J Biol Chem,
1990]
The nematode Caenorhabditis elegans (C. elegans) expresses the regulatory subunit (R) of cAMP-dependent protein kinase at a level similar to the levels determined for R subunits in mammalian tissues. Approximately 60% of the C. elegans cAMP-binding protein is tightly associated with particulate structures by noncovalent interactions. Ionic detergents or 7 M urea solubilize particulate R. Solubilized and cytosolic R subunits have apparent Mr values of 52,000 and pI values of 5.5. cDNA and genomic DNA encoding a unique C. elegans R subunit were cloned and sequenced. The derived amino acid sequence contains 375 residues; carboxyl-terminal residues 145-375 are 69% identical with mammalian RI. However, residues 44-145 are markedly divergent from the corresponding regions of all other R sequences. This region might provide sufficient structural diversity to adapt a single R subunit for multiple functional roles in C. elegans. Antibodies directed against two epitopes in the deduced amino acid sequence of C. elegans R avidly bound nematode cytosolic and particulate R subunits on Western blots and precipitated dissociated R subunits and R2C2 complexes from solution. Immunofluorescence analysis revealed that the tip of the head, which contains chemosensory and mechanosensory neurons, and the pharyngeal nerve ring were enriched in R. The R subunit concentration is low during early embryogenesis in C. elegans. A sharp increase (approximately 6-fold) in R content begins several hours before the nematodes hatch and peaks during the first larval stage. Developmental regulation of R expression occurs at translational and/or post-translational levels. The 8-kilobase pair C. elegans R gene is divided into 8 exons by introns ranging from 46 to 4300 base pairs. The 5'-flanking region has no TATA box and contains preferred and minor transcription start sites.
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[
Worm Breeder's Gazette,
1994]
R-ras I and R-ras 2 (TC21) homologs Per Winge*, Vercna Gobel*+, Stephen Friend*, and John Fleming*+. MGH Cancer Center and +DepL of Pediatrics, Boston, MA. Human r-ras 1 and r-ras 2 (TC21) belong to the closer relatives (>50% amino acid identity) of ras in the ras superfamily of GDP/GTP-binding proteins. They are the first members to exhibit transforming potential when mutated at some which render ras oncogenic and make it insensitive to GAP action (Graham & Der, 1994). These recent findings have led to current investigations of their role-in human cancer. Furthermore, r-ras 1 -- by immunoprecipitation and in the yeast-2-hybrid-system -- was shown to interact with
bc1-2, the human homolog to
ced-9 (Fernandez-Sarabia & Bischoff, 1993) and has thus been implicated as a possible effector of apoptosis. There is evidence that the r-ras proteins participate in some but not all aspects of the ras signal transduction pathway involving upstream tyrosinc kinases and downstream serine/threonine kinases. It has not yet been elucidated in the mammalian system (1) what alternative pathway the r-ras proteins may be utilizing and (2) what functional relevance is represented by the in vitro interaction of r-ras 1 and
bc1-2. We are trying to address these questions in C elegans and have cloned the homologs of r-ras I and r-ras 2 using a degeneratc PCR approach. We have screened c-DNA and genomic libraries and obtamed and sequenced full length c-DNA and genomic clones of r-ras 1 and a full length c-DNA clone of r- ras 2. The genomic sequence of r-ras 2 was recently made available by the genome sequencing project. The amino acid comparison shows high homologyrldentity to thc human proteins for r-ras 1 and r-ras 2 (TC21). R-ras 1 was localizcd to chromosome II ncar
lin-29, and r-ras 2 maps close to embS on chromosome m. To obtain r-ras germline deletions, we have screened a TCl insertion library which we constructed using the mutator strain MT 3126 (protocols kindly proYided by Jocl Rothman, Susan Mango and Ed Maryon), and have isolated transposon insertions in r-ras 1. We are currently in the proccss of sib sclection to purify the strains. To get some first appreciation of a functional role of r-ras towards apoptosis versus growth stimulating propertics, we have also started to inject a r-ras 1 hcat shock promotor expression construct to generatc strains in which r-ras can be overexpressed Ihis additional approach has been choscn since redundancy may be expected in thc ras related protcin familics and thus thc knockout of one of the proteins may not give clear results. We will screen the overexpressing strains for (1) apoptosis and (2) muv phcnotype. In collaboration with Bob Horvitz's laboratory r-ras GST fusion proteins will be generated to test the in vitro interacion with
ccd-9. Finally, we are constructing r-ras 1 and r-ras 2 promotor expression vectors with GFP/betaGAL to define the expression patterns of both genes.
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[
Nat Commun,
2021]
R-bodies are long, extendable protein polymers formed in the cytoplasm of some bacteria; they are best known for their role in killing of paramecia by bacterial endosymbionts. Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes (referred to as the reb cluster) with potential to confer production of R-bodies and that have been implicated in virulence. Here, we show that products of the PA14 reb cluster associate with R-bodies and control stochastic expression of R-body structural genes.PA14 expresses reb genes during colonization of plant and nematode hosts, and R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that P. aeruginosa R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. Our observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection.