Daulby J et al. (1996) European Worm Meeting "Glutathione-s-transferases of C.elegans"

Isaac RE, Daulby J, Coates D

[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.

Daulby J et al. (1998) European Worm Meeting "Characterisation of the sigma class glutathione S-transferases of C. elegans"

Isaac E, Daulby J, Coates D

[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

Daulby J et al. (1997) International C. elegans Meeting "CHARACTERISATION OF THE SIGMA CLASS OF GLUTATHIONE-S-TRANSFERASES (GSTs) OF C.ELEGANS."

Daulby J, Lynch AS, Isaac RE, Coates D

[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

J Daulby et al. (1999) International C. elegans Meeting "Characterisation of sigma class glutathione s-transferases of C.elegans"

D Coates, RE Isaac, J Daulby

[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

McGhee JD (1987) Biochemistry "Purification and characterization of a carboxylesterase from the intestine of the nematode Caenorhabditis elegans."

McGhee JD

[Biochemistry, 1987]

The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N- terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].

Murakami S et al. (1999) Curr Biol "Life extension and stress resistance in Caenorhabditis elegans modulated by the tkr-1 gene."

Johnson TE, Murakami S

[Curr Biol, 1999]

In this Brief Communication, which appeared in the 14 September 1998 issue of Current Biology, the UV dose was reported erroneously. The dose reported was 20 J/m2 but the actual dose used was 0.4 J/cm2. Also, the gene formally referred to as tkr-1 has since been renamed old-1 (overexpression longevity determination).

Ramos CG et al. (2014) J Bacteriol "Retraction for Ramos et al., MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia."

Feliciano JR, da Costa PJ, Ramos CG, Grilo AM, Leitao JH

[J Bacteriol, 2014]

Volume 195, no. 16, p. 35143523, 2013. A number of problems related to images published in this paper have been brought to our attention. Figure 1D contains duplicated images in lanes S and LE, and Fig. 4D and 6B contain images previously published in articles in this journal and in Microbiology and Microbial Pathogenesis, i.e., the following: C. G. Ramos, S. A. Sousa, A. M. Grilo, J. R. Feliciano, and J. H. Leitao, J. Bacteriol. 193:15151526, 2011. doi:10.1128/JB.01374-11. S. A. Sousa, C. G. Ramos, L. M. Moreira, and J. H. Leitao, Microbiology 156:896908, 2010. doi:10.1099/mic.0.035139-0. C. G. Ramos, S. A. Sousa, A. M. Grilo, L. Eberl, and J. H. Leitao, Microb. Pathog. 48:168177, 2010. doi: 10.1016/j.micpath.2010.02.006. Therefore, we retract the paper. We deeply regret this situation and apologize for any inconvenience to the editors and readers of Journal of Bacteriology, Microbial Pathogenesis, and Microbiology.

Nillegoda NB et al. (2015) Nature "Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation."

Berynskyy M, Morimoto RI, Bukau B, Stengel F, Kirstein J, Szlachcic A, Arnsburg K, Stank A, Scior A, Nillegoda NB, Gao X, Guilbride DL, Aebersold R, Wade RC, Mayer MP

[Nature, 2015]

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.

Coates D et al. (2000) European Worm Meeting "The ADAMs family of C. elegans: what will the new members do?"

Coates D, Daulby J, Hooper NM, Hope IA

[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.

Miller DM et al. (1992) Worm Breeder's Gazette "unc-4 LacZ Expression in A-Type Motor Neurons"

Miller DM, Niemeyer CJ

[Worm Breeder's Gazette, 1992]

unc-4 LacZ expression in A-type motor neurons David M. Miller and Charles J. Niemeyer, Dept. of Cell Biology, Duke Univ. Medical Ctr, Durham, NC 27710