Hapiak, Vera M. et al. (2011) International Worm Meeting "TYRA-3 mediates the tyraminergic "inhibition" of 5-HT-stimulated aversive responses in Caenorhabditis elegans."

Stein, A., Komuniecki, R. W., Korchnak, A., Hapiak, Vera M.

[International Worm Meeting, 2011]

Monoamines and peptides modulate neuronal plasticity and interact to define behavioral state in both vertebrates and invertebrates. In Caenorhabditis elegans, monoamines modulate aversive responses mediated by the polymodal, nociceptive ASH sensory neurons (Wragg et al., 2007; Harris et al., 2009, 2010). Previously, we have shown that food or 5-HT-stimulated aversive responses to dilute octanol are delayed by both octopamine (OA) and tyramine (TA) and demonstrated that the OA inhibition of ASH-mediated aversive responses is mediated by three different OA receptors that either modulate the ASHs directly or stimulate the release of an array of neuropeptides from the ADL, AWB and ASI sensory neurons that inhibit maximal ASH-mediated aversive responses through distinct peptide receptors expressed outside the ASH-mediated circuit (Wragg et al., 2010 C. elegans Neuro Meeting, #115). In the present study, we have dissected the role of TYRA-3, a Gaq-coupled TA receptor, in the TA-dependent delay of 5-HT-stimulated aversive responses to dilute octanol. TA does not inhibit 5-HT-stimulated responses in tyra-3 null animals and this TA "inhibition" can be fully restored by the expression of a full-length tyra-3 transgene. tyra-3 is expressed in the ventral ganglion, gonad, and a number of neurons in the head and tail, based on expression of full-length rescuing ptyra-3::tyra-3:gfp transgenes. Importantly, as described above for OA, the TA signal modulating ASH-mediated aversive behavior also appears to be amplified by the tyra-3-dependent release of neuropeptides that appear to differ significantly from those involved in OA-dependent "inhibition." These studies are continuing to define the relationship between monoaminergic and peptidergic signaling in the modulation of sensory-mediated locomotory behaviors.

Vera M. Hapiak et al. (2007) International Worm Meeting "TYRA-3 (M03F4.3) belongs to a novel family of invertebrate tyramine receptors."

Sarah B. Miller, Gareth P. Harris, Richard W. Komuniecki, Robert J. Hobson, Rachel T. Wragg, Vera M. Hapiak

[International Worm Meeting, 2007]

Tyramine (TA) regulates a number of key processes in nematodes, including pharyngeal pumping, egg-laying, and locomotion, suggesting that TA-mediated signaling may provide useful targets for anthelminthic development, as described previously for insect OA/TA receptors. Previously, we characterized two C. elegans G<font face=symbol>a</font>o-coupled TA receptors, SER-2 and TYRA-2. In the present study, we have identified a third C. elegans gene, tyra-3 (m03f4.3) that also encodes a TA receptor. TYRA-3 clusters most closely within a novel, recently described, family of TA-specific insect receptors that activate Ca++ dependent chloride channels after expression in Xenopus oocytes (Cazzamali et al., 2005). Membranes from COS-7 cells expressing TYRA-3 exhibit saturable [3H]LSD binding (Kd = 31.1 &#177; 4.7 nM) and, in inhibition binding assays, TA inhibits [3H]LSD binding with much higher affinity than other classical biogenic amines (IC50, 0.8 &#177; 0.01 <font face=symbol>m</font>M). TYRA-3 exhibits a unique pharmacological profile and, in contrast to SER-2 and TYRA-2, couples to TA-dependent increases (3-5 fold) in cAMP levels after transient expression in COS-7 cells (EC50, 0.62 &#177; 0.12 <font face=symbol>m</font>M). In contrast, OA has no effect on cAMP levels. tyra-3::gfp gene fusions exhibit an expression pattern that is markedly different from those reported for either tyra-2 or ser-2. tyra-3::gfp is robustly expressed in the CEP/ADE dopaminergic neurons and additional neurons in the head and tail, as well as the gonad and intestine. Interestingly, TYRA-3, but not SER-2 or TYRA-2, is essential for the TA-dependent abolition of 5-HT mediated increases in sensitivity to dilute octanol, implying a key role for TYRA-3 in olfactory modulation. However, the neuronal circuit and downstream signaling involved in TYRA-3-mediated modulation remains to be identified. These studies are continuing to examine the role of TYRA-3 in other TA-dependent phenotypes.

Wypijewska A et al. (2012) Biochemistry "7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity."

Wypijewska A, Darzynkiewicz E, Davis RE, Jemielity J, Bojarska E, Lukaszewicz M, Stepinski J

[Biochemistry, 2012]

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Takeishi*, Asuka et al. (2015) International Worm Meeting "AFD-specific receptor guanylyl cyclases can confer temperature responses onto diverse cell types."

Takeishi*, Asuka, Sengupta, Piali, Bell, Harold W., Hapiak, Vera M., Yu*, Yanxun V.

[International Worm Meeting, 2015]

C. elegans exhibits complex experience-dependent thermosensory behaviors on spatial thermal gradients. AFD has been identified as the major thermosensory neuron type, and AFD-ablated worms exhibit strong defects in thermotaxis behaviors. Measurements of temperature-evoked intracellular calcium dynamics and thermoreceptor current have shown that AFD responds to thermal variation at temperatures above a threshold temperature (T*) that is set by the animal's cultivation temperature (Tc). Thermotransduction in AFD is mediated by cGMP signaling, and requires the functions of the receptor guanylyl cyclases (rGCs), GCY-8, GCY-18 and GCY-23, the cGMP-dependent phosphodiesterase PDE-2, and the TAX-2 and TAX-4 cGMP-gated channels. However, the molecular mechanisms by which AFD senses temperature are still unknown.Previous studies have shown that many rGCs function as sensors for specific stimuli, suggesting that the AFD-expressed rGCs may themselves act as thermosensors. We found that misexpression of GCY-8, GCY-18 and GCY-23 in the non-thermosensory AWB and ASE chemosensory neurons is sufficient to confer warming-dependent changes in intracellular calcium (also see abstract by Yu, Takeishi et al). We further expressed each rGC singly or in combination in these neurons, and found that expression of GCY-23 alone confers temperature responses onto AWB and ASE in the physiological temperature range, whereas expression of GCY-18 confers responses to higher temperatures. Interestingly, in contrast to AFD, the T* of AWB and ASE misexpressing AFD-specific rGCs is not modulated by Tc, suggesting that the observed Tc -dependent plasticity in AFD thermosensory response threshold may be mediated by AFD-specific mechanisms. To ask whether misexpression in cell types lacking endogenous cGMP signaling pathways would also confer thermosensory responses, we next misexpressed the rGCs in vulval muscles. We found that vulval muscles misexpressing GCY-23 as well as TAX-2/TAX-4 also exhibit responses to temperature variation. Our results suggest that AFD-specific rGCs can confer thermosensory responses onto multiple neuronal and non-neuronal cell types, and raise the possibility that these proteins may be bona fide thermosensors in C. elegans.* - equal contributions.

O'Connell EM et al. (2015) J Infect Dis "Targeting Filarial Abl-like Kinases: Orally Available, Food and Drug Administration-Approved Tyrosine Kinase Inhibitors Are Microfilaricidal and Macrofilaricidal."

Nutman TB, O'Connell EM, Bennuru S, Steel C, Dolan MA

[J Infect Dis, 2015]

BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and &gt;100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.

Marks NJ et al. (1999) Biochem Biophys Res Commun "Isolation, pharmacology and gene organization of KPSFVRFamide: a neuropeptide from Caenorhabditis elegans."

Alexander-Bowman S, Verhaert P, Marks NJ, Maule AG, Li C, Halton DW, Thompson DP, Shaw C, Nelson LS, Geary TG

[Biochem Biophys Res Commun, 1999]

To date, 53 peptides with C-terminal RFamides have been identified by the genome sequencing project in the nematode, Caenorhabditis elegans. In this study the FMRFamide-related peptide (FaRP) KPSFVRFamide (879.90 Da [MH]+) was structurally characterized from extracts of the nematode, Caenorhabditis elegans. Two copies of KPSFVRFamide are encoded by a gene designated flp-9. RT-PCR identified a single cDNA product which was confirmed as flp-9 by sequence determination. Flp-9 cDNA was isolated from larval stages of C. elegans but was not detected in adult worms, indicating that its expression is may be developmentally regulated. KPSFVRFamide displays sequence homology to the nematode peptide, KPNFIRFamide (PF4). The physiological effects of KPSFVRFamide, PF4 and the chimeras, KPNFVRFamide and KPSFIRFamide, were measured on body wall muscle and the vagina vera of the parasitic nematode, Ascaris suum. KPNFVRFamide and KPNFIRFamide had Cl--dependent inhibitory activity on innervated and denervated muscle-preparations, whereas KPSFVRFamide and KPSFIRFamide did not elicit a detectable physiological effect. Although all 4 peptides had inhibitory effects on the vagina vera, KPSFVRFamide and KPSFIRFamide (threshold, >/=0.1 microM) were less potent than KPNFVRFamide and KPNFIRFamide (threshold, >/=10 nM).

Wood WB (1976) Worm Breeder's Gazette "maternal effects among ts zygote-defective (zyg) mutants."

Wood WB

[Worm Breeder's Gazette, 1976]

We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.

Ali Khan L et al. (1994) Worm Breeder's Gazette "cej-1 Encodes a Novel Protein With Poly-Threonine Motif"

Siddiqui SS, Fukushige T, Ali Khan L, Tsukita Sh, Tabish M, Itoh M

[Worm Breeder's Gazette, 1994]

cej-1 Encodes a Novel Protein with Poly-Threonine Motif M. L. A. Khanl, M. Tabish, T. Fukushigel1 S. Tsukita2, M. Itoh , Sh. Tsukita , and S. S. Siddiqui. (1): Lab. of Molecular Biology, Dept of Ecological Engg. Toyohashi Univ. Technology, Toyohashi 441, and (2). National Institute for Physiological Sciences, Okazaki 444, Japan.

Lemire BD et al. (2009) Mech Ageing Dev "C. elegans longevity pathways converge to decrease mitochondrial membrane potential."

Behrendt M, Decorby A, Lemire BD, Gaskova D

[Mech Ageing Dev, 2009]

Energy production via oxidative phosphorylation generates a mitochondrial membrane potential (DeltaPsi(m)) across the inner membrane. In this work, we show that a lower DeltaPsi(m) is associated with increased lifespan in Caenorhabditis elegans. The long-lived mutants daf-2(e1370), age-1(hx546), clk-1(qm30), isp-1(qm150) and eat-2(ad465) all have a lower DeltaPsi(m) than wild type animals. The lower DeltaPsi(m) of daf-2(e1370) is daf-16 dependent, indicating that the insulin-like signaling pathway not only regulates lifespan but also mitochondrial energetics. RNA interference (RNAi) against 17 genes shown to extend lifespan also decrease DeltaPsi(m). Furthermore, lifespan can be significantly extended with the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP), which dissipates DeltaPsi(m). We conclude that longevity pathways converge on the mitochondria and lead to a decreased DeltaPsi(m). Our results are consistent with the 'uncoupling to survive' hypothesis, which states that dissipation of the DeltaPsi(m) will extend lifespan.

Seo J et al. (2005) Arch Environ Contam Toxicol "Biodegradation of the insecticide N,N-diethyl-m-toluamide by fungi: identification and toxicity of metabolites."

Kim SD, Cha CJ, Hur HG, Lee YG, Seo J, Ahn JH

[Arch Environ Contam Toxicol, 2005]

Fungi (Cunninghamella elegans ATCC 9245, Mucor ramannianus R-56, Aspergillus niger VKMF-1119, and Phanerochaete chrysosporium BKMF-1767) were tested to elucidate the biologic fate of the topical insect repellent N,N-diethyl-m-toluamide (DEET). The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column, showed that three peaks occurred after incubation of C. elegans, with which 1 mM DEET was combined as a final concentration. The peaks were not detected in the control experiments with either DEET alone or tested fungus alone. The metabolites produced by C. elegans exhibited a molecular mass of 207 with a fragment ion (m/z) at 135, a molecular mass of 179 with an m/z at 135, and a molecular mass of 163 with an m/z at 119, all of which correspond to N,N-diethyl-m-toluamide-N-oxide, N-ethyl-m-toluamide-N-oxide, and N-ethyl-m-toluamide, respectively. M. ramannianus R-56 also produced N, N-diethyl-m-toluamide-N-oxide and N-ethyl-m-toluamide but did not produce N-ethyl-m-toluamide-N-oxide. For the biologic toxicity test with DEET and its metabolites, the freshwater zooplankton Daphnia magna was used. The biologic sensitivity in decreasing order was DEET > N-ethyl-m-toluamide > N,N-diethyl-m-toluamide-N-oxide. Although DEET and its fungal metabolites showed relatively low mortality compared with other insecticides, the toxicity was increased at longer exposure periods. These are the first reports of the metabolism of DEET by fungi and of the biologic toxicity of DEET and its fungal metabolites to the freshwater zooplankton D. magna.