RESUMEN
Originally designed as a general alternative to acute fish toxicity testing (AFT), the fish embryo toxicity test (FET) has become subject to concerns with respect to neurotoxic substances. Whereas oxygen uptake in the fish embryo primarily occurs via diffusion across the skin, juvenile and adult fish rely on active ventilation of the gills. As a consequence, substances including, e.g., neurotoxicants which prevent appropriate ventilation of gills ("respiratory failure syndrome") might lead to suffocation in juvenile and adult fish, but not in skin-breathing embryos. To investigate if this respiratory failure syndrome might play a role for the higher sensitivity of juvenile and adult fish to neurotoxicants, a modified acute toxicity test using post-embryonic, early gill-breathing life-stages of zebrafish was developed with chlorpyrifos, permethrin, lindane, aldicarb, ziram and aniline as test substances. Additionally, a comparative study into bioaccumulation of lipophilic substances with logKow > 3.5 and swimbladder deflation as potential side effects of the respiratory failure syndrome was performed with 4 d old skin-breathing and 12 d old gill-breathing zebrafish. With respect to acute toxicity, post-embryonic 12 d larvae proved to be more sensitive than both embryos (FET) and adult zebrafish (AFT) to all test substances except for permethrin. Accumulation of chlorpyrifos, lindane and permethrin was 1.3- to 5-fold higher in 4 d old than in 12 d old zebrafish, suggesting that (intermediate) storage of substances in the yolk might reduce bioavailability and prevent metabolization, which could be a further reason for lower toxicity in 4 d than in 12 d old zebrafish. Whereas ziram and aniline showed no significant effect on the swimbladder, zebrafish exposed to chlorpyrifos, lindane and permethrin showed significantly deflated swimbladders in 12 d old larvae; in the case of aldicarb, there was a significant hyperinflation in 4 d old larvae. Swimbladder deflation in post-embryonic 12 d zebrafish larvae might be hypothesized as a reason for a lack of internal oxygen supplies during the respiratory failure syndrome, whereas in 4 d old embryos cholinergic hyperinflation of the swimbladder dominates over other effects. Regarding acute lethality, the study provides further evidence that the switch from transcutaneous to branchial respiration in post-embryonic zebrafish life-stages might be the reason for the higher sensitivity of juvenile and adult fish to neurotoxic substances.
Asunto(s)
Cloropirifos , Insuficiencia Respiratoria , Contaminantes Químicos del Agua , Ziram , Aldicarb/farmacología , Compuestos de Anilina/farmacología , Animales , Cloropirifos/toxicidad , Embrión no Mamífero , Branquias , Hexaclorociclohexano , Larva , Oxígeno , Permetrina/farmacología , Respiración , Pruebas de Toxicidad Aguda/métodos , Contaminantes Químicos del Agua/toxicidad , Pez Cebra , Ziram/farmacologíaRESUMEN
Complex biological functions within organisms are frequently orchestrated by systemic communication between tissues. In the model organism Caenorhabditis elegans, the pharyngeal and body wall neuromuscular junctions are two discrete structures that control feeding and locomotion, respectively. Separate, the well-defined neuromuscular circuits control these distinct tissues. Nonetheless, the emergent behaviors, feeding and locomotion, are coordinated to guarantee the efficiency of food intake. Here, we show that pharmacological hyperactivation of cholinergic transmission at the body wall muscle reduces the rate of pumping behavior. This was evidenced by a systematic screening of the effect of the cholinesterase inhibitor aldicarb on the rate of pharyngeal pumping on food in mutant worms. The screening revealed that the key determinants of the inhibitory effect of aldicarb on pharyngeal pumping are located at the body wall neuromuscular junction. In fact, the selective stimulation of the body wall muscle receptors with the agonist levamisole inhibited pumping in a lev-1-dependent fashion. Interestingly, this response was independent of unc-38, an alpha subunit of the nicotinic receptor classically expressed with lev-1 at the body wall muscle. This implies an uncharacterized lev-1-containing receptor underpins this effect. Overall, our results reveal that body wall cholinergic transmission not only controls locomotion but simultaneously inhibits feeding behavior.
Asunto(s)
Proteínas de Caenorhabditis elegans , Inhibidores de la Colinesterasa , Conducta Alimentaria , Unión Neuromuscular , Aldicarb/farmacología , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Inhibidores de la Colinesterasa/farmacología , Conducta Alimentaria/efectos de los fármacos , Conducta Alimentaria/fisiología , Levamisol/farmacología , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Transducción de SeñalRESUMEN
Inhibition of acetylcholinesterase by either organophosphates or carbamates causes anti-cholinesterase poisoning. This arises through a wide range of neurotoxic effects triggered by the overstimulation of the cholinergic receptors at synapses and neuromuscular junctions. Without intervention, this poisoning can lead to profound toxic effects, including death, and the incomplete efficacy of the current treatments, particularly for oxime-insensitive agents, provokes the need to find better antidotes. Here we show how the non-parasitic nematode Caenorhabditis elegans offers an excellent tool for investigating the acetylcholinesterase intoxication. The C. elegans neuromuscular junctions show a high degree of molecular and functional conservation with the cholinergic transmission that operates in the autonomic, central and neuromuscular synapses in mammals. In fact, the anti-cholinesterase intoxication of the worm's body wall neuromuscular junction has been unprecedented in understanding molecular determinants of cholinergic function in nematodes and other organisms. We extend the use of the model organism's feeding behaviour as a tool to investigate carbamate and organophosphate mode of action. We show that inhibition of the cholinergic-dependent rhythmic pumping of the pharyngeal muscle correlates with the inhibition of the acetylcholinesterase activity caused by aldicarb, paraoxons and DFP exposure. Further, this bio-assay allows one to address oxime dependent reversal of cholinesterase inhibition in the context of whole organism recovery. Interestingly, the recovery of the pharyngeal function after such anti-cholinesterase poisoning represents a sensitive and easily quantifiable phenotype that is indicative of the spontaneous recovery or irreversible modification of the worm acetylcholinesterase after inhibition. These observations highlight the pharynx of C. elegans as a new tractable approach to explore anti-cholinesterase intoxication and recovery with the potential to resolve critical genetic determinants of these neurotoxins' mode of action.
Asunto(s)
Antídotos/uso terapéutico , Bioensayo/métodos , Caenorhabditis elegans/efectos de los fármacos , Inhibidores de la Colinesterasa/envenenamiento , Faringe/efectos de los fármacos , Aldicarb/farmacología , Animales , Intoxicación por Organofosfatos/diagnóstico , Faringe/fisiologíaRESUMEN
Ion channels are present at specific levels within subcellular compartments of excitable cells. The regulation of ion channel trafficking and targeting is an effective way to control cell excitability. The BK channel is a calcium-activated potassium channel that serves as a negative feedback mechanism at presynaptic axon terminals and sites of muscle excitation. The C. elegans BK channel ortholog, SLO-1, requires an endoplasmic reticulum (ER) membrane protein for efficient anterograde transport to these locations. Here, we found that, in the absence of this ER membrane protein, SLO-1 channels that are seemingly normally folded and expressed at physiological levels undergo SEL-11/HRD1-mediated ER-associated degradation (ERAD). This SLO-1 degradation is also indirectly regulated by a SKN-1A/NRF1-mediated transcriptional mechanism that controls proteasome levels. Therefore, our data indicate that SLO-1 channel density is regulated by the competitive balance between the efficiency of ER trafficking machinery and the capacity of ERAD.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Proteínas de Unión al ADN/metabolismo , Degradación Asociada con el Retículo Endoplásmico/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Terminales Presinápticos/metabolismo , Factores de Transcripción/metabolismo , Aldicarb/farmacología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Acoplamiento Excitación-Contracción/efectos de los fármacos , Acoplamiento Excitación-Contracción/genética , Retroalimentación Fisiológica/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Músculos/inervación , Terminales Presinápticos/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal , Isoformas de Proteínas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The ACC-1 family of cys-loop receptors are ligand-gated chloride channels sensitive to acetylcholine (ACh), and are only present in invertebrates. Studies of this family of inhibitory receptors has provided insight into how they bind and respond to ACh in a manner vastly different from nicotinic acetylcholine receptors and appear to be present in tissues that are relevant to anthelmintic action. Here, we have identified two members of the ACC-1 family from the parasitic nematode Haemonchus contortus, Hco-LGC-46 and Hco-ACC-4. Hco-LGC-46 is an ACC subunit that has never been previously expressed and pharmacologically characterized. We found that Hco-LGC-46 when expressed in Xenopus laevis oocytes forms a functional homomeric channel that is responsive to the cholinergic agonists ACh and methylcholine. hco-lgc-46 expressed in a C. elegans lgc-46 null strain (ok2900) suppressed hypersensitivity to aldicarb in a manner similar to cel-lgc-46. It was also found that Hco-LGC-46 assembles with Hco-ACC-1 and produces a receptor that is over 5-fold more sensitive to ACh and responds to the cholinergic agonists methycholine and carbachol. In contrast, the co-expression of Hco-LGC-46 with Hco-ACC-4 resulted in non-functional channels in oocytes. Hco-ACC-4 also appears to form heteromeric channels with a previously characterized subunit, Hco-ACC-2. Co-expression of Hco-ACC-4 with Hco-ACC-2 resulted in a functional heteromeric channel with an EC50 value similar to that of the Hco-ACC-2 homomeric channel. However, the maximum currents generated in the ACC-4/ACC-2 channel were significantly (p < 0.005) lower than those from the ACC-2 homomeric channel. Overall, this is the first report confirming that lgc-46 encodes an acetylcholine-gated chloride channel which when co-expressed with acc-4 results in reduced receptor function or trafficking in oocytes.
Asunto(s)
Acetilcolina/metabolismo , Canales de Cloruro/química , Receptores de Canales Iónicos con Asa de Cisteína Activados por Ligando/química , Haemonchus/metabolismo , Proteínas del Helminto/química , Acetilcolina/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Aldicarb/farmacología , Secuencia de Aminoácidos , Animales , Antihelmínticos/farmacología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Carbacol/metabolismo , Carbacol/farmacología , Canales de Cloruro/genética , Canales de Cloruro/aislamiento & purificación , Canales de Cloruro/metabolismo , Colina/análogos & derivados , Colina/metabolismo , Colina/farmacología , Clonación Molecular , Receptores de Canales Iónicos con Asa de Cisteína Activados por Ligando/genética , Receptores de Canales Iónicos con Asa de Cisteína Activados por Ligando/aislamiento & purificación , Receptores de Canales Iónicos con Asa de Cisteína Activados por Ligando/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Haemonchus/genética , Proteínas del Helminto/genética , Proteínas del Helminto/aislamiento & purificación , Proteínas del Helminto/metabolismo , Modelos Moleculares , Oocitos/citología , Oocitos/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
Inhibitory GABAergic transmission is required for proper circuit function in the nervous system. However, our understanding of molecular mechanisms that preferentially influence GABAergic transmission, particularly presynaptic mechanisms, remains limited. We previously reported that the ubiquitin ligase EEL-1 preferentially regulates GABAergic presynaptic transmission. To further explore how EEL-1 functions, here we performed affinity purification proteomics using Caenorhabditis elegans and identified the O-GlcNAc transferase OGT-1 as an EEL-1 binding protein. This observation was intriguing, as we know little about how OGT-1 affects neuron function. Using C. elegans biochemistry, we confirmed that the OGT-1/EEL-1 complex forms in neurons in vivo and showed that the human orthologs, OGT and HUWE1, also bind in cell culture. We observed that, like EEL-1, OGT-1 is expressed in GABAergic motor neurons, localizes to GABAergic presynaptic terminals, and functions cell-autonomously to regulate GABA neuron function. Results with catalytically inactive point mutants indicated that OGT-1 glycosyltransferase activity is dispensable for GABA neuron function. Consistent with OGT-1 and EEL-1 forming a complex, genetic results using automated, behavioral pharmacology assays showed that ogt-1 and eel-1 act in parallel to regulate GABA neuron function. These findings demonstrate that OGT-1 and EEL-1 form a conserved signaling complex and function together to affect GABA neuron function.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Neuronas GABAérgicas/fisiología , N-Acetilglucosaminiltransferasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Aldicarb/farmacología , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/aislamiento & purificación , Cromatografía de Afinidad , Neuronas GABAérgicas/efectos de los fármacos , Terminales Presinápticos/metabolismo , Unión Proteica , Proteómica , Transducción de Señal , Transmisión Sináptica/efectos de los fármacos , Ubiquitina-Proteína Ligasas/aislamiento & purificaciónRESUMEN
The gut microbiome is highly involved in numerous aspects of host physiology, from energy harvest to stress response, and can confer many benefits to the host. The gut microbiome development could be affected by genetic and environmental factors, including pesticides. The carbamate insecticide aldicarb has been extensively used in agriculture, which raises serious public health concerns. However, the impact of aldicarb on the gut microbiome, host metabolome, and lipidome has not been well studied yet. Herein, we use multiomics approaches, including16S rRNA sequencing, shotgun metagenomics sequencing, metabolomics, and lipidomics, to elucidate aldicarb-induced toxicity in the gut microbiome and the host metabolic homeostasis. We demonstrated that aldicarb perturbed the gut microbiome development trajectory, enhanced gut bacterial pathogenicity, altered complex lipid profile, and induced oxidative stress, protein degradation, and DNA damage. The brain metabolism was also disturbed by the aldicarb exposure. These findings may provide a novel understanding of the toxicity of carbamate insecticides.
Asunto(s)
Aldicarb/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Insecticidas/farmacología , Lípidos , Metaboloma/efectos de los fármacos , Administración Oral , Aldicarb/administración & dosificación , Animales , Daño del ADN , Insecticidas/administración & dosificación , Lipidómica , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Estrés Oxidativo/efectos de los fármacosRESUMEN
The aberrant regulation of Wnt secretion is implicated in various neurological diseases. However, the mechanisms of Wnt release are still largely unknown. Here we describe the role of a C. elegans tetraspan protein, HIC-1, in maintaining normal Wnt release. We show that HIC-1 is expressed in cholinergic synapses and that mutants in hic-1 show increased levels of the acetylcholine receptor AChR/ACR-16. Our results suggest that HIC-1 maintains normal AChR/ACR-16 levels by regulating normal Wnt release from presynaptic neurons, as hic-1 mutants show an increase in secreted Wnt from cholinergic neurons. We further show that HIC-1 affects Wnt secretion by modulating the actin cytoskeleton through its interaction with the actin-binding protein NAB-1. In summary, we describe a protein, HIC-1, that functions as a neuromodulator by affecting postsynaptic AChR/ACR-16 levels by regulating presynaptic Wnt release from cholinergic motor neurons.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Wnt/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Aldicarb/farmacología , Animales , Caenorhabditis elegans/efectos de los fármacos , Proteínas de Caenorhabditis elegans/química , Neuronas Colinérgicas/efectos de los fármacos , Neuronas Colinérgicas/metabolismo , Proteínas de la Membrana/química , Proteínas de Microfilamentos/química , Músculos/efectos de los fármacos , Proteínas Mutantes/metabolismo , Mutación/genética , Proteínas del Tejido Nervioso/química , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/metabolismoRESUMEN
The role of Notch signaling in cell-fate decisions has been studied extensively; however, this pathway is also active in adult tissues, including the nervous system. Notch signaling modulates a wide range of behaviors and processes of the nervous system in the nematode Caenorhabditis elegans, but there is no evidence for Notch signaling directly altering synaptic strength. Here, we demonstrate Notch-mediated regulation of synaptic activity at the C. elegans neuromuscular junction (NMJ). For this, we used aldicarb, an inhibitor of the enzyme acetylcholinesterase, and assessed paralysis rates of animals with altered Notch signaling. Notch receptors LIN-12 and GLP-1 are required for normal NMJ function; they regulate NMJ activity in an opposing fashion. Complete loss of LIN-12 skews the excitation/inhibition balance at the NMJ toward increased activity, whereas partial loss of GLP-1 has the opposite effect. Specific Notch ligands and co-ligands are also required for proper NMJ function. The role of LIN-12 is independent of cell-fate decisions; manipulation of LIN-12 signaling through RNAi knockdown or overexpression of the co-ligand OSM-11 after development alters NMJ activity. We demonstrate that LIN-12 modulates GABA signaling in this paradigm, as loss of GABA signaling suppresses LIN-12 gain-of-function defects. Further analysis, in vivo and in silico, suggests that LIN-12 may modulate transcription of the GABAB receptor GBB-2 Our findings confirm a non-developmental role for the LIN-12/Notch receptor in regulating synaptic signaling and identify the GABAB receptor GBB-2 as a potential Notch transcriptional target in the C. elegans nervous system.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Unión Neuromuscular/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Ácido gamma-Aminobutírico/metabolismo , Aldicarb/farmacología , Animales , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Inhibidores de la Colinesterasa/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación , Unión Neuromuscular/efectos de los fármacos , Receptores Notch/genética , Transducción de Señal/efectos de los fármacosRESUMEN
The bionematicidal effect of a synthetic volatile mixture (SVM) of four volatile organic compounds (VOCs) emitted by the endophytic fungus Daldinia cf. concentrica against the devastating plant-parasitic root-knot nematode Meloidogyne javanica has been recently demonstrated in both in vitro and greenhouse experiments. However, the mode of action governing the observed irreversible paralysis of J2 larvae upon exposure to SVM is unknown. To unravel the mechanism underlying the anthelmintic and nematicidal activities, we used the tractable model worm Caenorhabditis elegans. C. elegans was also susceptible to both the fungal VOCs and SVM. Among compounds comprising SVM, 3-methyl-1-butanol, (±)-2-methyl-1-butanol, and 4-heptanone showed significant nematicidal activity toward L1, L4 and young adult stages. Egg hatching was only negatively affected by 4-heptanone. To determine the mechanism underlying this activity, we examined the response of C. elegans mutants for glutamate-gated chloride channel and acetylcholine transporter, targets of the nematicidal drugs ivermectin and aldicarb, respectively, to 4-heptanone and SVM. These aldicarb- and ivermectin-resistant mutants retained susceptibility upon exposure to 4-heptanone and SVM. Next, we used C. elegans TJ356 strain zIs356 (daf-16::GFP+rol-6), LD1 ldIs7 [skn-1B/C::GFP + pRF4(rol-6(su1006))], LD1171 ldIs3 [gcs-1p::gfp; rol-6(su1006))], CL2166 dvIs19 (gst-4p::GFP) and CF1553 muIs84 (sod-3p::GFP+rol-6), which have mutations in genes regulating multiple stress responses. Following exposure of L4 larvae to 4-heptanone or SVM, there was clear nuclear translocation of DAF-16::GFP, and SKN-1::GFP indicating that their susceptibility involves DAF-16 and SKN1 regulation. Application of 4-heptanone, but not SVM, induced increased expression of, gcs-1::GFP and gst-4::GFP compared to controls. In contrast, application of 4-heptanone or SVM to the sod-3::GFP line elicited a significant decline in overall fluorescence intensity compared to controls, indicating SOD-3 downregulation and therefore overall reduction in cellular redox machinery. Our data indicate that the mode of action of SVM and 4-heptanone from D. cf. concentrica differs from that of currently available nematicides, potentially offering new solutions for nematode management.
Asunto(s)
Antihelmínticos/farmacología , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/efectos de los fármacos , Factores de Transcripción Forkhead/genética , Larva/efectos de los fármacos , Compuestos Orgánicos Volátiles/farmacología , Xylariales/química , Aldicarb/farmacología , Animales , Antihelmínticos/aislamiento & purificación , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/agonistas , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción Forkhead/agonistas , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Ivermectina/farmacología , Cetonas/química , Cetonas/farmacología , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Pentanoles/química , Pentanoles/farmacología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Compuestos Orgánicos Volátiles/aislamiento & purificación , Xylariales/metabolismo , Cigoto/efectos de los fármacos , Cigoto/crecimiento & desarrollo , Cigoto/metabolismoRESUMEN
The heterotrimeric G protein Gq regulates neuronal activity through distinct downstream effector pathways. In addition to the canonical Gq effector phospholipase Cß, the small GTPase Rho was recently identified as a conserved effector of Gq. To identify additional molecules important for Gq signaling in neurons, we performed a forward genetic screen in the nematode Caenorhabditis elegans for suppressors of the hyperactivity and exaggerated waveform of an activated Gq mutant. We isolated two mutations affecting the MAP kinase scaffold protein KSR-1 and found that KSR-1 modulates locomotion downstream of, or in parallel to, the Gq-Rho pathway. Through epistasis experiments, we found that the core ERK MAPK cascade is required for Gq-Rho regulation of locomotion, but that the canonical ERK activator LET-60/Ras may not be required. Through neuron-specific rescue experiments, we found that the ERK pathway functions in head acetylcholine neurons to control Gq-dependent locomotion. Additionally, expression of activated LIN-45/Raf in head acetylcholine neurons is sufficient to cause an exaggerated waveform phenotype and hypersensitivity to the acetylcholinesterase inhibitor aldicarb, similar to an activated Gq mutant. Taken together, our results suggest that the ERK MAPK pathway modulates the output of Gq-Rho signaling to control locomotion behavior in C. elegans.
Asunto(s)
Caenorhabditis elegans/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Locomoción , Sistema de Señalización de MAP Quinasas , Proteínas de Unión al GTP rho/metabolismo , Aldicarb/farmacología , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Neuronas Colinérgicas/efectos de los fármacos , Neuronas Colinérgicas/metabolismo , Neuronas Colinérgicas/fisiología , Inhibidores de la Colinesterasa/farmacología , Epistasis Genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Quinasas raf/genética , Quinasas raf/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
This study is the first to report sound production in Japanese medaka (Oryzias latipes). Sound production was affected by exposure to the carbamate insecticide (aldicarb) and heavy-metal compound (copper sulfate). Medaka were exposed at four concentrations (aldicarb: 0, 0.25, 0.5, and 1 mg L-1; copper sulfate: 0, 0.5, 1, and 2 mg L-1), and sound characteristics were monitored for 5 h after exposure. We observed constant average interpulse intervals (approx 0.2 s) in all test groups before exposure, and in the control groups throughout the experiment. The average interpulse interval became significantly longer during the recording periods after 50 min of exposure to aldicarb, and reached a length of more than 0.3 s during the recording periods after 120 min exposure. Most medaka fish stopped to produce sound after 50 min of exposure to copper sulfate at 1 and 2 mg L-1, resulting in significantly declined number of sound pulses and pulse groups. Relative shortened interpulse intervals of sound were occasionally observed in medaka fish exposed to 0.5 mg L-1 copper sulfate. These alternations in sound characteristics due to toxicants exposure suggested that they might impair acoustic communication of medaka fish, which may be important for their reproduction and survival. Our results suggested that using acoustic changes of medaka has potential to monitor precipitate water pollutions, such as intentional poisoning or accidental leakage of industrial waste.
Asunto(s)
Aldicarb/farmacología , Sulfato de Cobre/farmacología , Oryzias/fisiología , Sonido , Animales , Monitoreo del Ambiente/métodos , Insecticidas/farmacología , Reproducción , Contaminantes Químicos del Agua/farmacologíaRESUMEN
Parasitic nematodes negatively impact human and animal health worldwide. The market withdrawal of nematicidal agents due to unfavourable toxicities has limited the available treatment options. In principle, co-administering nematicides at lower doses along with molecules that potentiate their activity could mitigate adverse toxicities without compromising efficacy. Here, we screened for new small molecules that interact with aldicarb, which is a highly effective treatment for plant-parasitic nematodes whose toxicity hampers its utility. From our collection of 638 worm-bioactive compounds, we identified 20 molecules that interact positively with aldicarb to either kill or arrest the growth of the model nematode Caenorhabditis elegans. We investigated the mechanism of interaction between aldicarb and one of these novel nematicides called wact-86. We found that the carboxylesterase enzyme GES-1 hydrolyzes wact-86, and that the interaction is manifested by aldicarb's inhibition of wact-86's metabolism by GES-1. This work demonstrates the utility of C. elegans as a platform to search for new molecules that can positively interact with industrial nematicides, and provides proof-of-concept for prospective discovery efforts.
Asunto(s)
Aldicarb/farmacología , Antinematodos/farmacología , Benzamidas/farmacología , Benzofuranos/farmacología , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/efectos de los fármacos , Hidrolasas de Éster Carboxílico/genética , Nematodos/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antinematodos/química , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Mutación , Alineación de SecuenciaRESUMEN
One of the key issues pertaining to the control of memory is to respond to a consistently changing environment or microbial niche present in it. Human cyclic AMP response element binding protein (CREB) transcription factor which plays a crucial role in memory has a homolog in C. elegans, crh-1. crh-1 appears to influence memory processes to certain extent by habituation of the host to a particular environment. The discrimination between the pathogen and a non-pathogen is essential for C. elegans in a microbial niche which determines its survival. Training the nematodes in the presence of a virulent pathogen (S. aureus) and an opportunistic pathogen (P. mirabilis) separately exhibits a different behavioural paradigm. This appears to be dependent on the CREB transcription factor. Here we show that C. elegans homolog crh-1 helps in memory response for a short term against the interacting pathogens. Following conditioning of the nematodes to S. aureus and P. mirabilis, the wild type nematodes exhibited a positive response towards the respective pathogens which diminished slowly after 2h. By contrast, the crh-1 deficient nematodes had a defective memory post conditioning. The molecular data reinforces the importance of crh-1 gene in retaining the memory of nematode. Our results also suggest that involvement of neurotransmitters play a crucial role in modulating the memory of the nematode with the assistance of CREB. Therefore, we elucidate that CREB is responsible for the short term memory response in C. elegans against bacterial pathogens.
Asunto(s)
Bacterias/inmunología , Caenorhabditis elegans/microbiología , Caenorhabditis elegans/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Interacciones Huésped-Patógeno/inmunología , Memoria Inmunológica , Aldicarb/farmacología , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Humanos , Memoria Inmunológica/genética , Mutación , Fosforilación , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The precise role of huntingtin-associated protein 1 (HAP1) is not known, but studies have shown that it is important for early development and survival. A Caenorhabditis elegans ortholog of HAP1, T27A3.1 (also called trak-1), has been found and is expressed in a subset of neurons. Potential behavioral functions of three knockout lines of T27A3.1 were examined. From its suspected role in mice we hypothesize that T27A3.1 might be involved in egg hatching and early growth, mechanosensation, chemosensation, sensitivity to osmolarity, and synaptic transmission. Our studies show that the knockout worms are significantly different from the wild-type (WT) worms only in the synaptic transmission test, which was measured by adding aldicarb, an acetylcholinesterase inhibitor. The change in function was determined by measuring the number of worms paralyzed. However, when the T27A3.1 worms were tested for egg hatching and early growth, mechanosensation, chemosensation, and sensitivity to osmolarity, there were no significant differences between the knockout and WT worms. © 2016 Wiley Periodicals, Inc.
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Conducta Animal , Proteínas de Caenorhabditis elegans/genética , Proteínas del Tejido Nervioso/genética , Aldicarb/farmacología , Animales , Caenorhabditis elegans , Quimiotaxis/genética , Inhibidores de la Colinesterasa/farmacología , Técnicas de Inactivación de Genes , Actividad Motora , Concentración Osmolar , Reproducción/efectos de los fármacos , Sensación , Sinapsis , Transmisión SinápticaRESUMEN
The highly conserved cochaperone DnaJ/Hsp40 family proteins are known to interact with molecular chaperone Hsp70, and can regulate many cellular processes including protein folding, translocation, and degradation. In studies of Caenorhabditis elegans locomotion mutants, we identified a gain-of-function (gf) mutation in dnj-17 closely linked to the widely used e156 null allele of C. elegans GAD (glutamic acid decarboxylase) unc-25 dnj-17 encodes a DnaJ protein orthologous to human DNAJA5. In C. elegans DNJ-17 is a cytosolic protein and is broadly expressed in many tissues. dnj-17(gf) causes a single amino acid substitution in a conserved domain, and behaves as a hypermorphic mutation. The effect of this dnj-17(gf) is most prominent in mutants lacking GABA synaptic transmission. In a seizure model caused by a mutation in the ionotropic acetylcholine receptor acr-2(gf), dnj-17(gf) exacerbates the convulsion phenotype in conjunction with absence of GABA. Null mutants of dnj-17 show mild resistance to aldicarb, while dnj-17(gf) is hypersensitive. These results highlight the importance of DnaJ proteins in regulation of C. elegans locomotor circuit, and provide insights into the in vivo roles of DnaJ proteins in humans.
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Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas del Choque Térmico HSP40/genética , Convulsiones/genética , Transmisión Sináptica/genética , Aldicarb/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Conducta Animal , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Inhibidores de la Colinesterasa/farmacología , Secuencia Conservada , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Proteínas del Choque Térmico HSP40/deficiencia , Proteínas del Choque Térmico HSP40/metabolismo , Humanos , Locomoción , Receptores Nicotínicos/deficiencia , Receptores Nicotínicos/genética , Convulsiones/metabolismo , Convulsiones/fisiopatología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Ácido gamma-Aminobutírico/deficienciaRESUMEN
Previously, we identified 25 classifier genes that were able to assess immunotoxicity using human Jurkat T cells. The present study aimed to validate these classifiers. For that purpose, Jurkat cells were exposed for 6 h to subcytotoxic doses of nine immunotoxicants, five non-immunotoxicants and four compounds for which human immunotoxicity has not yet been fully established. RNA was isolated and subjected to Fluidigm quantitative real time (qRT)-PCR analysis. The sensitivity, specificity and accuracy of the screening assay as based on the nine immunotoxicants and five non-immunotoxicants used in this study were 100%, 80% and 93%, respectively, which is better than the performance in our previous study. Only one compound was classified as false positive (benzo-e-pyrene). Of the four potential (non-)immunotoxicants, chlorantraniliprole and Hidrasec were classified immunotoxic and Sunset yellow and imidacloprid as non-immunotoxic. ToxPi analysis of the PCR data provided insight in the molecular pathways that were affected by the compounds. The immunotoxicants 2,3-dichloro-propanol and cypermethrin, although structurally different, affected protein metabolism and cholesterol biosynthesis and transport. In addition, four compounds, i.e. chlorpyrifos, aldicarb, benzo-e-pyrene and anti-CD3, affected genes in cholesterol metabolism and transport, protein metabolism and transcription regulation. qRT-PCR on eight additional genes coding for similar processes as defined in ToxPi analyzes, supported these results. In conclusion, the 25 immunotoxic classifiers performed very well in a screening with new non-immunotoxic and immunotoxic compounds. Therefore, the Jurkat screening assay has great promise to be applied within a tiered approach for animal free testing of human immunotoxicity.
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Marcadores Genéticos/efectos de los fármacos , Inmunotoxinas/farmacología , Células Jurkat/efectos de los fármacos , Aldicarb/farmacología , Aldicarb/toxicidad , Compuestos Azo/farmacología , Compuestos Azo/toxicidad , Benzopirenos/farmacología , Benzopirenos/toxicidad , Biomarcadores Farmacológicos , Clorhidrinas/farmacología , Clorhidrinas/toxicidad , Cloropirifos/farmacología , Cloropirifos/toxicidad , Humanos , Imidazoles/farmacología , Imidazoles/toxicidad , Técnicas In Vitro , Neonicotinoides , Nitrocompuestos/farmacología , Nitrocompuestos/toxicidad , Piretrinas/farmacología , Piretrinas/toxicidad , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Pruebas de Toxicidad , ortoaminobenzoatos/farmacología , ortoaminobenzoatos/toxicidadRESUMEN
The L1CAM family of cell adhesion molecules is a conserved set of single-pass transmembrane proteins that play diverse roles required for proper nervous system development and function. Mutations in L1CAMs can cause the neurological L1 syndrome and are associated with autism and neuropsychiatric disorders. L1CAM expression in the mature nervous system suggests additional functions besides the well-characterized developmental roles. In this study, we demonstrate that the gene encoding the Caenorhabditis elegans L1CAM, sax-7, genetically interacts with gtl-2, as well as with unc-13 and rab-3, genes that function in neurotransmission. These sax-7 genetic interactions result in synthetic phenotypes that are consistent with abnormal synaptic function. Using an inducible sax-7 expression system and pharmacological reagents that interfere with cholinergic transmission, we uncovered a previously uncharacterized nondevelopmental role for sax-7 that impinges on synaptic function.
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Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/genética , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Sinapsis/metabolismo , Aldicarb/farmacología , Animales , Animales Modificados Genéticamente , Medios de Cultivo , Epistasis Genética , Expresión Génica , Mutación , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/genética , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genéticaRESUMEN
The complex molecular and cellular mechanisms underlying neuronal control of animal movement are not well understood. Locomotion of Caenorhabditis elegans is mediated by a neuronal circuit that produces coordinated sinusoidal movement. Here we utilize this simple, yet elegant, behaviour to show that VAV-1, a conserved guanine nucleotide exchange factor for Rho-family GTPases, negatively regulates motor circuit activity and the rate of locomotion. While vav-1 is expressed in a small subset of neurons, we find that VAV-1 function is required in a single interneuron, ALA, to regulate motor neuron circuit activity. Furthermore, we show by genetic and optogenetic manipulation of ALA that VAV-1 is required for the excitation and activation of this neuron. We find that ALA signalling inhibits command interneuron activity by abrogating excitatory signalling in the command interneurons, which is responsible for promoting motor neuron circuit activity. Together, our data describe a novel neuromodulatory role for VAV-1-dependent signalling in the regulation of motor circuit activity and locomotion.
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Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Interneuronas/metabolismo , Locomoción/fisiología , Actividad Motora/fisiología , Proteínas Proto-Oncogénicas c-vav/genética , Aldicarb/farmacología , Animales , Animales Modificados Genéticamente , Proteínas de Caenorhabditis elegans/biosíntesis , Agonistas Colinérgicos/farmacología , Inhibidores de la Colinesterasa/farmacología , Antagonistas del GABA/farmacología , Levamisol/farmacología , Locomoción/genética , Actividad Motora/genética , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Parálisis/inducido químicamente , Pentilenotetrazol/farmacología , Proteínas Proto-Oncogénicas c-vav/biosíntesis , Interferencia de ARN , ARN Interferente Pequeño , Receptores Colinérgicos , Factores de Intercambio de Guanina Nucleótido Rho , Rodopsina/biosíntesis , Transducción de SeñalRESUMEN
Alzheimer's disease (AD) is the most common and devastating neurodegenerative disease. The etiology of AD has yet to be fully understood, and common treatments remain largely non-efficacious. The amyloid hypothesis posits that extracellular amyloid-ß (Aß) deposits are the fundamental etiological factor of the disease. The present study tested the organoselenium compound diphenyl-diselenide (PhSe)2, which is characterized by its antioxidant and antiinflammatory properties and has shown efficacy in several neurodegenerative disease models. We employed a transgenic Caenorhabditis elegans AD model to analyze the effects of (PhSe)2 treatment on Aß peptide-induced toxicity. Chronic exposure to (PhSe)2 attenuated oxidative stress induced by Aß1-42, with concomitant recovery of associative learning memory in C. elegans. Additionally, (PhSe)2 decreased Aß1-42 transgene expression, suppressed Aß1-42 peptide, and downregulated hsp-16.2 by reducing the need for this chaperone under Aß1-42-induced toxicity. These observations suggest that (PhSe)2 plays an important role in protecting against oxidative stress-induced toxicity, thus representing a promising pharmaceutical modality that attenuates Aß1-42 expression.