Chitwood DJ et al. (1987) Comparative Biochemistry and Physiology "Sterol metabolism in the nematodes Panagrellus redivivus, Turbatrix aceti and C. elegans."

Chitwood DJ, Salt TA, Lusby WR

[Comparative Biochemistry and Physiology, 1987]

1. Panagrellus redivivus, Turbatrix aceti and Caenorhabditis elegans were sterilely propagated in semidefined media containing sitosterol or cholesterol, and sterols were isolated and identifed by capillary gas-liquid chromatography-mass spectometry. 2. Each species was capable of removal of the C-24 ethyl substituent of sitosterol and production of 4a-methylsterols. 3. Other modifications of the sterol nucleus varied among the species, as only T. aceti and C. elegans introduced *7- and *8(14)-bonds significantly. 4. P. redivivus and, to a much lesser extent, T. aceti reduced *5-bonds to produce substantial quantities of cholesterol and 4a-methylcholestanol.

Gerrits D et al. (1996) European Worm Meeting "TYROSINASES OF CAENORHABDITIS ELEGANS"

Gerrits D, Blaxter ML

[European Worm Meeting, 1996]

The cuticle of C. elegans is extensively cross-linked by covalent disulphide bridges, tyrosine bonds and possibly glutamyl-lysine bonds. Two enzymes presumably involved in the formation of tyrosine bonds have been identified in C. elegans. Both genes map to chromosome III. tyr-1 and tyr-2 appear to be very similar in structure: both genes have two Cu active sites (CuA and CuB) and two NC6 domains (found in other proteins from C. elegans and Toxocara canis ). tyr-1 has an additional polyglutamine region which may be involved in protein-protein interactions. A set of cDNA's prepared from a synchronous population of worms, harvested at two hour intervals through the lifecycle, starting shortly after hatching (cDNA kindly provided by I. Johnstone), was used in semi-quantitative fluorescent PCR. Preliminary results suggest that both genes are upregulated at each moult, suggesting their involvement in the synthesis of the new cuticle. Different parts of both genes have been cloned and expressed in E. coli. Antibodies against these recombinant proteins were raised in mice and will be used to locate the native proteins in worms.

Alan Winter et al. (2008) European Worm Meeting "Elucidation of collagen specific roles for the multifunctional ER folding enzyme protein disulphide isomerase."

Antony Page, Alan Winter, Gillian Mccormack

[European Worm Meeting, 2008]

The multifunctional enzyme protein disulphide isomerase (PDI) is required. for many aspects of protein folding and transit through the endoplasmic. reticulum. Secretory pathway proteins are in an unfolded state as they. enter the endoplasmic reticulum (ER) where a number resident enzymes and. chaperones are present to ensure correct folding. Proteins in the secretory. pathway are frequently stabilised by disulphide bonds which cross-link. specific cysteine residues. Protein folding is inhibited both by the. formation of incorrect disulphide bonds and by formation of bonds in the. wrong temporal sequence. Additionally spontaneous folding of disulphide. containing proteins occurs at an extremely slow rate. PDI catalyses native. disulphide bonding by introducing disulphide bonds (oxidase activity) and. through breakage and rearrangement of incorrect disulphide bonds (isomerase. activity).. PDI has been implicated in collagen biosynthesis in multi-cellular. animals primarily through its presence in the prolyl 4-hydroxylase (P4H). enzyme complex. P4H catalyses the conversion of proline residues to 4-. hydroxyproline in collagen Gly-X-Pro repeats. This modified residue is. required for the thermal stability of mature collagen with the enzymes. responsible being essential for survival, morphogenesis and formation of. the cuticular extra-cellular matrix (ECM) in C. elegans. Cross-linking of. conserved cysteine residues of collagens suggests PDI may also have a P4H-. independent role in collagen biogenesis.. We have functionally analysed, particularly with respect to ECM. formation, three C. elegans PDI enzymes, PDI-1, -2 and -3, using genetic. mutants. Mutation in pdi-2 results in severe body morphology defects,. uncoordinated movement, adult sterility, abnormal moulting and aberrant. collagen deposition. These phenotypes are consistent with a role in. collagen biogenesis and ECM formation. Site-directed mutagenesis and. transgenic re-expression demonstrates the importance of the catalytic. activity of PDI-2 indicating a P4H independent role. PDI-1 and PDI-3 are. individually non-essential, however, compound mutants of pdi-2 and -3 are. temperature sensitive embryonic lethal. All three genes are required for. completion of embryonic development at all temperatures. The function of. PDI-2 is conserved in parasitic nematodes, where transgenic repair of the. C. elegans mutant can be achieved through expression of a PDI from the. human infective filarial nematode Brugia malayi. The essential conserved. role of PDI-2 makes this an attractive drug target for control of parasitic. nematodes.

Wang C et al. (2018) Chem Sci "Reductive cleavage of C[double bond, length as m-dash]C bonds as a new strategy for turn-on dual fluorescence in effective sensing of H2S."

Zhang X, Ding Z, Li G, Zhang H, Yuan D, Wang C, Tan J, Wang W, Cheng X

[Chem Sci, 2018]

Reductive cleavage of alkenes is rarely reported in synthetic chemistry. Here we report a unique H₂S-mediated reductive cleavage of C[double bond, length as m-dash]C bonds under mild conditions, which is a successful new strategy for the design of probes for effective sensing of H₂S with turn-on dual-color fluorescence. A short series of phenothiazine ethylidene malononitrile derivatives were shown to react with H₂S, <i>via</i> reductive cleavage of C[double bond, length as m-dash]C bonds with intramolecular cyclization reactions to form thiophene rings. Enlightened by this new reaction mechanism, four effective probes with turn-off to turn-on fluorescence switches were successfully applied for sensing H₂S, an important gaseous signalling molecule in living systems, among which PTZ-P4 exhibited two fluorescent colors after reductive cleavage. The dual-color probe was applied for imaging endogenous H₂S and showed distinct differences in brightness in living <i>C. elegans</i> for wild type N2, <i>glp-1</i> (<i>e2144</i>) mutants (higher levels of endogenous H₂S), and <i>cth-1</i> (<i>ok3319</i>) mutants (lower levels of endogenous H₂S). The discovery of H₂S-mediated reductive cleavage of C[double bond, length as m-dash]C bonds is expected to be valuable for chemical synthesis, theoretical studies, and the design of new fluorescent H₂S probes.

Shi L et al. (2018) Nat Commun "Optical imaging of metabolic dynamics in animals."

Zhang L, Chen Z, Silveira ES, Targoff K, Shi L, Zheng C, Min W, de Sena-Tomas C, Shen Y, Wei M, Liu C

[Nat Commun, 2018]

Direct visualization of metabolic dynamics in living animals with high spatial and temporal resolution is essential to understanding many biological processes. Here we introduce a platform that combines deuterium oxide (D₂O) probing with stimulated Raman scattering (DO-SRS) microscopy to image in situ metabolic activities. Enzymatic incorporation of D₂O-derived deuterium into macromolecules generates carbon-deuterium (C-D) bonds, which track biosynthesis in tissues and can be imaged by SRS in situ. Within the broad vibrational spectra of C-D bonds, we discover lipid-, protein-, and DNA-specific Raman shifts and develop spectral unmixing methods to obtain C-D signals with macromolecular selectivity. DO-SRS microscopy enables us to probe de novo lipogenesis in animals, image protein biosynthesis without tissue bias, and simultaneously visualize lipid and protein metabolism and reveal their different dynamics. DO-SRS microscopy, being noninvasive, universally applicable, and cost-effective, can be adapted to a broad range of biological systems to study development, tissue homeostasis, aging, and tumor heterogeneity.

Cox GN et al. (1981) J Cell Biol "Cuticle of Caenorhabditis elegans: its isolation and partial characterization."

Edgar RS, Kusch M, Cox GN

[J Cell Biol, 1981]

The adult cuticle of the soil nematode, Caenorhabditis elegans, is a proteinaceous extracellular structure elaborated by the underlying layer of hypodermal cells during the final molt in the animal's life cycle. The cuticle is composed of an outer cortical layer connected by regularly arranged struts to an inner basal layer. The cuticle can be isolated largely intact and free of all cellular material by sonication and treatment with 1% sodium dodecyl sulfate (SDS). Purified cuticles exhibit a negative material in the basal cuticle layer. The cuticle layers differ in their solubility in sulfhydryl reducing agents, susceptibility to various proteolytic enzymes and amino acid composition. The struts, basal layer, and internal cortical layer are composed of collagen proteins that are extensively cross-linked by disulfide bonds. The external cortical layer appears to contain primarily noncollagen proteins that are extensively cross- linked by nonreducible covalent bonds. The collagen proteins extracted from the cuticle with a reducing agent can be separated by SDS-polyacrylamide gel electrophoresis into eight major species differing in apparent molecular weight.

Wilson WR et al. (1994) Mol Biochem Parasitol "The Onchocerca volvulus homologue of the multifunctional polypeptide protein disulfide isomerase."

Shepley KJ, Tuan RS, Greene BM, Wilson WR, Unnasch TR, Freedman DO, Awadzi K

[Mol Biochem Parasitol, 1994]

Protein disulfide isomerase (PDI) functions to catalyze the formation of correct disulfide bonds in nascent proteins, and also acts as one of the subunits of prolyl-4 hydroxylase, the enzyme responsible for the oxidative maturation of procollagen. Since the cuticle of parasitic nematodes consists primarily of a network of collagen molecules which are connected through intermolecular disulfide bonds, PDI might be expected to be involved in the process of cuticle biosynthesis. The isolation and characterization of a cDNA encoding the PDI homologue of Onchocerca volvulus is described. This cDNA contains a single, long open reading frame that encodes sequence motifs identical to the two known active sites of PDI for isomerase activity. The O. volvulus PDI appears to be encoded by a single copy gene. Both in situ hybridization and immunolocalization data suggest that PDI is both spatially and temporally regulated in O. volvulus. The pattern of spatial and temporal regulation is consistent with the involvement of PDI in the biosynthesis of the parasite cuticle. The parasite protein appears to be an antigen recognized by a minority of individuals exposed to O. volvulus.

Cox GN (1992) Journal of Parasitology "Molecular and biochemical aspects of nematode collagens."

Cox GN

[Journal of Parasitology, 1992]

Collagens are major structural proteins of nematode cuticles and basement membranes (basal laminae). The collagen proteins that form these structures differ in their biochemical and physical properties and are encoded by distinct gene families. Nematode basement membrane collagens are large proteins that show strong homology to basement membrane collagens of vertebrates. There appear to be 2 nonidentical basement membrane collagen genes in nematodes. Cuticle collagens are about one-sixth the size of basement membrane collagens and are encoded by a large family of 20-150 nonidentical genes. Cuticle collagens can be subdivided into 4 families based upon certain structural features in the proteins. The mature, extracellular forms of both types of collagen proteins are extensively cross-linked by disulfide bonds and are largely insoluble in the absence of a thiol-reducing agent. Cuticle collagens are also cross-linked by nonreducible covalent bonds that involve tyrosine residues. The experimental studies that have led to our current understanding of the structures of basement membrane and cuticle collagens are reviewed. Some previous questions about the physical properties of these proteins are reexamined in light of the primary sequence information now available for the

Aamna Kaul et al. (2003) International Worm Meeting "Hyperactivation of glutamate-gated chloride channels with ivermectin results in necrosis."

Joseph Dent, Aamna Kaul

[International Worm Meeting, 2003]

Ivermectin is a widely used antiparasitic drug. It kills worms by activating glutamate-gated chloride channels (GluCls), which belong to the family of ligand-gated anion channels that includes the GABA and glutamate receptors (Cully et al., 1994; Dent et al., 2000). The chloride permeability that ivermectin induces in excitable cells tends to prevent excitation. For example, ivermectin targets a GluCl expressed in the pharyngeal muscle to inhibit muscle contraction and prevent eating (Dent et al., 1997). The worms linger for several days in the presence of ivermectin before they starve to death. However, we have found that the lethal effects of ivermectin on C. elegans become irreversible after only a few hours of exposure. When L1 worms were exposed to 20ng/ml for 5 hours and then washed, they gradually developed large vacuoles in their pharyngeal muscle over the next several days. A mutant strain that lacks ivermectin receptors shows little or no necrosis when treated. Ivermectin is hydrophobic and it irreversibly opens GluCls expressed in Xenopus oocytes. So it is possible that ivermectin persists in membranes and continues to activate GluCls. Furthermore, it has been shown that hyperactive cation channels can induce excitotoxic necrosis (Driscoll and Chalfie, 1991). Why, though, would an inhibitory channel have a similar effect when hyperactivated? We are trying to address this question by looking at whether mutations known to inhibit excitotoxicity also inhibit the necrotic effects of ivermectin. Cully DF, Vassilatis DK, Liu KK, Paress PS, Van der Ploeg LHT, Schaeffer JM, Arena JP. Nature 371: 707-711 1994 Dent JA, Smith MM, Vassilatis DK, Avery L. PNAS USA 97: 2674-2679 2000 Dent JA, Davis MW, Avery L. EMBO Journal 16: 5867-5879 1997 Driscoll, M and Chalfie, M. Nature 349: 588-593 1991

Conraths FJ et al. (1997) Exp Parasitol "Expression of the microfilarial sheath protein 2 (shp2) of the filarial parasites Litomosoides sigmodontis and Brugia malayi."

Hobom G, Zahner H, Conraths FJ, Hirzmann J

[Exp Parasitol, 1997]

The microfilarial sheaths of the filarial parasites Brugia malayi, Brugia pahangi, and Litomosoides sigmodontis consist of several parasite proteins, probably ranging between 7 and 10. The gene encoding sheath protein 2 (shp2), which is the object of this study, is transcribed in embryos and in the uterine epithelium; at least in B. malayi, it is translated in both tissues. Apparently, shp2 is synthesized as a monomer, exported by the respective cells, and integrated into the microfilarial sheath. In the sheath, it exists as a highly polymerized molecule cross-linked by cysteine formation and other covalent bonds, presumably epsilon-(gamma-glutamyl)-lysine links.