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[
International C. elegans Meeting,
1995]
Neuropeptides are essential for the coordination of growth, development, homeostasis, reproduction and behaviour in invertebrates. Although only a small number of peptides have been isolated from nematodes, it is clear from immunocytochemical studies that the nervous system of nematodes are endowed with a variety of peptidergic neurons. A putative receptor gene (ZK643.3/4) has been sequenced as part of the C.elegans genome sequencing project. It is predicted to have seven TM domains, and a G- protein binding site, has sequence identity within TM 3, TM6 and TM7 with members of the secretin/calcitonin/PTH sub-class of receptors and can be classed as a new member of this receptor family. Expression pattern studies using a fusion gene comprised of regulatory sequences of ZK643.3/4 and lac Z in C.elegans indicate that the receptor is localised to the muscle cells that control the opening and closing of the vulva and to muscle cells in the head region of adult worms. This pattern of gene expression is consistent with a role for ZK643.3/4 and a CGRP -like peptide in the control of head movement and of egg-laying. Full length cDNA was obtained by RT-PCR amplification. The 5' region of the transcript was identified using RT-PCR with an SL1 primer and an exon 1 primer, and confirmed using RNAse protection. Sequencing of the full length cDNA has highlighted the need to confirm gene structures predicted in genome sequencing projects by analysis of cDNAs. Comparison with the sequences of other members of the same sub-class of receptors has identified residues which are conserved, both within the transmembrane domains, and in several of the cytoplasmic loops. There is particularly high homology between the insect DH receptor and ZK643.3/4 in the third cytoplasmic loop.
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[
European Worm Meeting,
2000]
ADAMs are a family of integral membrane glycoproteins containing a disintegrin and a metalloprotease domain. The physiological roles of the majority of ADAMs have yet to be elucidated, however some mammalian ADAMs are known to be involved in many diverse processes including sperm migration, sperm-egg binding and fusion, myoblast fusion, the processing of adhesion molecules, cytokines, cytokine receptors and extracellular protein domains, and in neural development. In C. elegans ADAMs are thought to be involved in cell fusion events in sperm and in epithelial cells (ADM-1), in early embryonic development (ADM-2) and in vulval development (SUP-17). In addition to these membrane anchored proteins C. elegans also has soluble ADAM-like proteases, for example GON-1, which plays an essential role in gonadal morphogenesis. We have identified four novel ADAM-like sequences in C. eleganswhich encode a TNFa converting enzyme (TACE) homologue and three soluble ADAM-like proteinases, and our aim is to determine the precise functions of these proteins. Initially we will examine their cellular location with reporter gene constructs and assess the importance of these proteinases by generating gene knockout mutants. We are also interested in finding out what the substrates of these ADAMs are and will biochemically characterise the recombinant proteins. The data we will attain will be related to the ADAM homologues found in humans.
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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
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[
European Worm Meeting,
1998]
Termination of neurotransmitter-mediated signalling at the synapse is mainly carried out by members of the sodium- and chloride- coupled neurotransmitter transporter protein (NTP) family, which take neurotransmitter into presynaptic and glial cells by linking it to a sodium concentration gradient. They have a putative structure of 12 transmembrane domains linked by hydrophilic loops. To date over 40 members of the NTP family have been cloned and charaterised mainly from verterbrates. T25B6.7 was predicted to encode a member of the NTP family by the genome sequencing consortium. The promoter driven LacZ expression pattern of T25B6.7 is observed in embryonic, larval and adult stages. In the adult hermaphrodite staining is seen in neuronal processes in the phryngeal region, in lateral neuronal processes, in the vulval D cells, in the anal sphincter, and nuclei posterior to the anus. The adult male has similar pattern, staining is also visible in the copulatory apparatus. In the L1 stage, expression occurs in nuclei along the lateral aspect of the worm and in the ~400 min. embryonic stage there are nuclei staining in the dorso-lateral aspect in the posterior of the embryo. The larval and embryonic patterns of expression suggest the V and T lineages some of which go on to form neurones, socket cells and sheath cells. Deletions were carried out in the T25B6.7::LacZ construct to investigate promoter/enhancer elements. A deletion leaving 796bp of 5! flanking sequence reduced the level of expression of the T25B6::LacZ construct. Sequence upstream of this site is homologuous to 5! flanking sequence of other genes in the cosmids F21G4 and R03G8. A full length cDNA for this gene has been isolated and sequence analysis reveals that this is an atypical member of the family. Secondary structure prediction programs suggest an 11 transmembrane domain protein flanked by long carboxy and amino termini. Double stranded RNA was transcribed from the cDNA and injected into C.elegans, this produced late embryonic arrest. Vesicles were visible in the arrested embryo. The expression pattern and RNA experiments suggest that T25B6.7 is important in developmental stages of C.elegans and may be involved in blast cell fate inductions. It is difficult to speculate on the role of the gene in adult stages as its DNA sequence and expression pattern offers no obvious clues.
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Arai, Hiroyuki, Furuchi, Takemitsu, Okutsu, Mari, Homma, Hiroshi, Saitoh, Yasuaki, Katane, Masumi, Inoue, Takao, Sekine, Masae, Sakamoto, Taro
[
International Worm Meeting,
2013]
Among free D-amino acids existing in living organisms, D-serine (D-Ser) and D-aspartate (D-Asp) are the most intensively studied. In mammals, D-Ser has been proposed as a neuromodulator that regulates L-glutamate (L-Glu)-mediated activation of the N-methyl-D-Asp (NMDA) receptor by acting as a co-agonist. On the other hand, several lines of evidence suggest that D-Asp plays important roles in regulating hormone secretion and steroidogenesis. D-Amino acid oxidase (DAO) and D-Asp oxidase (DDO) are known as stereospecific degradative enzymes that catalyze the oxidative deamination of D-amino acids. Mammalian DAO and DDO are presumed to regulate endogenous D-Ser and D-Asp levels, respectively. Previously, we demonstrated that D-Ser, D-Asp, D-Glu and D-alanine (D-Ala) are present in nematode Caenorhabditis elegans, a multicellular model animal. We also found that C. elegans has at least one active DAO gene and three active DDO genes (DDO-1, DDO-2 and DDO-3), and that the spatiotemporal distributions of these enzymes in the body of C. elegans differ from one another. Furthermore, our previous study showed that alterations of brood size and hatching rate are observed in four C. elegans mutants lacking each gene for the DAO and DDOs. Interestingly, lifespan extension was observed in the DDO-3 mutant. To characterize the mechanism of lifespan extension in the DDO-3 mutant, we performed genetic epistasis experiments to test interactions between the DDO-3 gene and other known longevity pathways. The results suggest that DDO-3 is involved in caloric restriction-induced lifespan extension but not in insulin/IGF signaling pathway, NAD/sir2 pathway nor mitochondrial electron transport system. We also found that D-Glu and L-tryptophan (L-Trp) accumulate throughout life in the DDO-3 mutant. Now we are investigating the relationship between aging and the accumulations of D-Glu and L-Trp.
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Yousuke Seida, Masumi Katane, Hiroyuki Kobuna, Hiroyuki Arai, Masae Sekine, Hiroshi Homma, Kazuhiro Maeda, Taro Sakamoto, Yasuhito Nakagawa, Tomonori Kawata, Takemitsu Furuchi, Takao Inoue, Yasuaki Saitoh
[
East Asia Worm Meeting,
2010]
Among free D-amino acids existing in living organisms, D-serine (D-Ser) and D-aspartate (D-Asp) are the most actively studied. D-Ser has been proposed as a neuromodulator that regulates L-glutamate-mediated activation of the N-methyl-D-Asp (NMDA) receptor by acting as a co-agonist. On the other hand, several lines of evidence suggest that D-Asp plays important roles in regulating developmental processes, hormone secretion and steroidogenesis. D-Amino acid oxidase (DAO) and D-Asp oxidase (DDO) are known as stereospecific degradative enzymes that catalyze the oxidative deamination of D-amino acids. DAO displays broad substrate specificity and acts on several neutral and basic D-amino acids, while DDO is highly specific for acidic D-amino acids. DAO and DDO are presumed to regulate endogenous D-Ser and D-Asp levels, respectively, as well as mediate the elimination of accumulated exogenous D-amino acids in various organs. Previously, we demonstrated that nematode Caenorhabditis elegans, a multicellular model animal has at least one active DAO gene and three active DDO genes, while it had been thought that most organisms bear only one copy of each DAO and DDO gene. In addition, our previous study revealed that the spatiotemporal distributions of these enzymes in the body of C. elegans are different from one another. In this study, to elucidate the physiological roles of the C. elegans DAO and DDOs, we characterized several phenotypes of four C. elegans mutants in which each gene is partially deleted and inactivated. We also determined free D-amino acid contents in several worm samples using high-performance liquid chromatography (HPLC) techniques. We will report the phenotypes of the C. elegans mutants in comparison with those of wild-type C. elegans, as well as alterations in D-amino acid levels within the body.
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[
European Worm Meeting,
1996]
Glutathione-s-transferases (GSTs) are a large family of multifunctional enzymes involved in the detoxification and excretion of many physiological and xenobiotic substances in the cell. We are investigating the roles members of the sigma class of these enzymes may have in C.elegans in relation to their elimination of oxygen free radical damage which might contribute to cellular ageing. Initially we are examining expression pattern data in C.elegans lines transformed with LacZ reporter gene constructs containing predicted promoter regions of GST gene homologues. The predicted GST gene sequences were obtained from the genome sequencing project. All stages of development are being studied under a variety of environmental contditions to investigate GST gene expression inducibility, however particular attention will be paid to dauer stage larvae.
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[
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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[
International Worm Meeting,
2021]
Microbiome-derived metabolites are able to impact the host's nervous system through the gut-brain axis. D-amino acids (D-AAs), the D-enantiomers of prevalent L-amino acids, have unique functions such as neuromodulators in animals including mollusks, rodents, and primates, while microbiota is a known contributor to the source of D-AAs in animals. Among all D-AAs, D-Ala is a potent agonist of the glycine binding site of N-methyl-D-aspartate (NMDA) glutamate receptors in vitro. Additionally, D-Ala immunoreactivity in pancreatic beta-cells and adrenocorticotropic hormone (ACTH)-secreting cells suggests its involvement in glucose homeostasis. While several researches showed microbiota as a major source of D-Ala in animals, the effect of bacterial D-Ala synthesis and metabolism on host physiology has not been studied. In this study, we fed wild type Caenorhabditis elegans N2 with Escherichia coli mutants with knockout of genes relevant to D-Ala biosynthesis (dadX, alr) and D-Ala metabolism (ddlA), and then examined changes in C. elegans phenotypes with or without the presence of high glucose. All three bacterial mutants displayed similar growth curves in nutrient-rich liquid media, but decreased D-Ala/total alanine ratio compared to the parental E. coli strain. To prevent the introduction of exogenous D-Ala, we cultured C. elegans on peptone-free NGM media. We found no statistically significant differences in life span when fed on the selected E. coli mutants. However, we observed a slight avoidance to all three bacterial mutants compared to the parental strain. When amended with 40 mM glucose, deltaddlA significantly decreased the life span of N2. No food preference was observed on glucose-amended plates. These results indicate that the combination of deficiency in bacterial DdlA activity and high glucose led to a decreased life span in C. elegans.
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[
International C. elegans Meeting,
1997]
Glutathione-s-transferases (GSTs) are a large family of multifunctional enzymes principally involved in the detoxification and excretion of many physiological and xenobiotic substances. They protect the cell against the harmful effects of oxidative damage and have been implicated in retarding the ageing process. We are investigating the possible functions of members of the sigma (*) class of GST in C.elegans. A number of GST homologues have been identified by ACeDB and we have utilised this information to create upstream promoter region::LacZ fusion constructs. Strains transformed with the R03D7.6:LacZ construct exhibit expression in all post-embryonic stages of development in the vulval, pharyngeal and tail regions. However strong expression is seen in the excretory cell of the adult, suggesting a possible role of these GSTs in detoxification processes. Currently we are analysing GFP reporter data for R03D7.6 and examining the effects of oxidative stress on the expression of this gene. We have also obtained the full length R03D7.6 cDNA which will be used in antibody staining experiments and protein expression assays