Tribendimidine: Mode of Action and nAChR Subtype Selectivity in Ascaris and Oesophagostomum

The cholinergic class of anthelmintic drugs is used for the control of parasitic nematodes. One of this class of drugs, tribendimidine (a symmetrical diamidine derivative, of amidantel), was developed in China for use in humans in the mid-1980s. It has a broader-spectrum anthelmintic action against soil-transmitted helminthiasis than other cholinergic anthelmintics, and is effective against hookworm, pinworms, roundworms, and Strongyloides and flatworm of humans. Although molecular studies on C. elegans suggest that tribendimidine is a cholinergic agonist that is selective for the same nematode muscle nAChR as levamisole, no direct electrophysiological observations in nematode parasites have been made to test this hypothesis. Also the hypothesis that levamisole and tribendimine act on the same receptor, does not explain why tribendimidine is effective against some nematode parasites when levamisole is not. Here we examine the effects of tribendimidine on the electrophysiology and contraction of Ascaris suum body muscle and show that tribendimidine produces depolarization antagonized by the nicotinic antagonist mecamylamine, and that tribendimidine is an agonist of muscle nAChRs of parasitic nematodes. Further pharmacological characterization of the nAChRs activated by tribendimidine in our Ascaris muscle contraction assay shows that tribendimidine is not selective for the same receptor subtypes as levamisole, and that tribendimidine is more selective for the B-subtype than the L-subtype of nAChR. In addition, larval migration inhibition assays with levamisole-resistant Oesophagostomum dentatum isolates show that tribendimidine is as active on a levamisole-resistant isolate as on a levamisole-sensitive isolate, suggesting that the selectivity for levamisole and tribendimidine is not the same. It is concluded that tribendimidine can activate a different population of nematode parasite nAChRs than levamisole, and is more like bephenium. The different nAChR subtype selectivity of tribendimidine may explain why the spectrum of action of tribendimidine is different to that of other cholinergic anthelmintics like levamisole.     

Electrophysiological recording from parasitic nematode muscle

Infection of man and animals with parasitic nematodes is recognized as a significant global problem (McLeod in Int J Parasitol 25(11):1363–1367, 1994; Hotez et al. in N Engl J Med 357(10):1018–1027, 2007). At present control of these infections relies primarily on chemotherapy. There are a limited number of classes of anthelmintic compounds and the majority of these act on ion-channels of the parasite (Martin et al. in Parasitology 113:S137–S156, 1996). In this report, we describe electrophysiological recording techniques as applied to parasitic nematodes. The aim of this report is: (1) to promote the study of ion channels in nematodes to help further the understanding of antinematodal drug action; (2) to describe our recording equipment and experimental protocols; and (3) provide some examples of the information to be gleaned from this approach and how it can increase our understanding of these important pathogens.     

Cycloartane Triterpenes from Dikamali,the Gum Resin of Gardenia gummifera and Gardenia lucida

We report on the chemical investigation of dikamali gum, which is the resin of Gardenia gummifera and G. lucida (Rubiaceae). Six new cycloartane triterpenes, dikamaliartanes A–F ( 1 – 6 , resp.), together with a known flavonoid ( 7 ), were isolated and identified by NMR spectroscopy. All six cycloartanes are characterized by an open A‐ring with a free COOH group at C(3). In four of them, the C‐atoms C(23)–C(27) form a 4‐methylfuran‐2‐yl moiety. Bacterial assays using Staphylococcus aureus, Candida albicans, and Mycobacteria have been carried out but did not reveal significant activity.     

A behavioral switch: cGMP and PKC signaling in olfactory neurons reverses odor preference in C. elegans

Innate chemosensory preferences are often encoded by sensory neurons that are specialized for attractive or avoidance behaviors. Here, we show that one olfactory neuron in Caenorhabditis elegans, AWC(ON), has the potential to direct both attraction and repulsion. Attraction, the typical AWC(ON) behavior, requires a receptor-like guanylate cyclase GCY-28 that acts in adults and localizes to AWC(ON) axons. gcy-28 mutants avoid AWC(ON)-sensed odors; they have normal odor-evoked calcium responses in AWC(ON) but reversed turning biases in odor gradients. In addition to gcy-28, a diacylglycerol/protein kinase C pathway that regulates neurotransmission switches AWC(ON) odor preferences. A behavioral switch in AWC(ON) may be part of normal olfactory plasticity, as odor conditioning can induce odor avoidance in wild-type animals. Genetic interactions, acute rescue, and calcium imaging suggest that the behavioral reversal results from presynaptic changes in AWC(ON). These results suggest that alternative modes of neurotransmission can couple one sensory neuron to opposite behavioral outputs.     

Developmental wave of Brn3b expression leading to RGC fate specification is synergistically maintained by miR‐23a and miR‐374

Differential regulation of Brn3b is essential for the Retinal Ganglion Cell (RGC) development in the two phases of retinal histogenesis. This biphasic Brn3b regulation is required first, during early retinal histogenesis for RGC fate specification and secondly, during late histogenesis, where Brn3b is needed for RGC axon guidance and survival. Here, we have looked into how the regulation of Brn3b at these two stages happens. We identified two miRNAs, miR‐23a and miR‐374, as regulators of Brn3b expression, during the early stage of RGC development. Temporal expression pattern of miR‐23a during E10–19, PN1–7, and adult retina revealed an inverse relation with Brn3b expression. Though miR‐374 did not show such a pattern, its co‐expression with miR‐23a evidently inhibited Brn3b. We further substantiated these findings by ex vivo overexpression of these miRNAs in E14 mice retina and found that miR‐23a and miR‐374 together brings about a change in Brn3b expression pattern in ganglion cell layer (GCL) of the developing retina. From our results, it appears that the combined expression of these miRNAs could be regulating the timing of the wave of Brn3b expression required for early ganglion cell fate specification and later for its survival and maturation into RGCs. Taken together, here we provide convincing evidences for the existence of a co‐ordinated mechanism by miRNAs to down regulate Brn3b that will ultimately regulate the development of RGCs from their precursors. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1155–1171, 2014     

Neural network features distinguish chemosensory stimuli in Caenorhabditis elegans

Nervous systems extract and process information from the environment to alter animal behavior and physiology. Despite progress in understanding how different stimuli are represented by changes in neuronal activity, less is known about how they affect broader neural network properties. We developed a framework for using graph-theoretic features of neural network activity to predict ecologically relevant stimulus properties, in particular stimulus identity. We used the transparent nematode, Caenorhabditis elegans, with its small nervous system to define neural network features associated with various chemosensory stimuli. We first immobilized animals using a microfluidic device and exposed their noses to chemical stimuli while monitoring changes in neural activity of more than 50 neurons in the head region. We found that graph-theoretic features, which capture patterns of interactions between neurons, are modulated by stimulus identity. Further, we show that a simple machine learning classifier trained using graph-theoretic features alone, or in combination with neural activity features, can accurately predict salt stimulus. Moreover, by focusing on putative causal interactions between neurons, the graph-theoretic features were almost twice as predictive as the neural activity features. These results reveal that stimulus identity modulates the broad, network-level organization of the nervous system, and that graph theory can be used to characterize these changes.     

Oligomerization propensity and flexibility of yeast frataxin studied by X-ray crystallography and small-angle X-ray scattering

Frataxin is a mitochondrial protein with a central role in iron homeostasis. Defects in frataxin function lead to Friedreich's ataxia, a progressive neurodegenerative disease with childhood onset. The function of frataxin has been shown to be closely associated with its ability to form oligomeric species; however, the factors controlling oligomerization and the types of oligomers present in solution are a matter of debate. Using small-angle X-ray scattering, we found that Co²⁺, glycerol, and a single amino acid substitution at the N-terminus, Y73A, facilitate oligomerization of yeast frataxin, resulting in a dynamic equilibrium between monomers, dimers, trimers, hexamers, and higher-order oligomers. Using X-ray crystallography, we found that Co²⁺ binds inside the channel at the 3-fold axis of the trimer, which suggests that the metal has an oligomer-stabilizing role. The results reveal the types of oligomers present in solution and support our earlier suggestions that the trimer is the main building block of yeast frataxin oligomers. They also indicate that different mechanisms may control oligomer stability and oligomerization in vivo.     

Advantages of Repeated Low Dose against Single High Dose of Kainate in C57BL/6J Mouse Model of Status Epilepticus: Behavioral and Electroencephalographic Studies

A refined kainate (KA) C57BL/6J mouse model of status epilepticus (SE) using a repeated low dose (RLD) of KA (5 mg/kg, intraperitoneal; at 30 min intervals) was compared with the established single high dose (SHD) of KA (20 mg/kg, intraperitoneal) model. In the RLD group, increased duration of convulsive motor seizures (CMS, Racine scale stage ≥3) with a significant reduction in mortality from 21% to 6% and decreased variability in seizure severity between animals/batches were observed when compared to the SHD group. There was a significant increase in the percentage of animals that reached stage-5 seizures (65% versus 96%) in the RLD group. Integrated real-time video-EEG analysis of both groups, using NeuroScore software, revealed stage-specific spikes and power spectral density characteristics. When the seizures progressed from non-convulsive seizures (NCS, stage 1–2) to CMS (stage 3–5), the delta power decreased which was followed by an increase in gamma and beta power. A transient increase in alpha and sigma power marked the transition from NCS to CMS with characteristic ‘high frequency trigger’ spikes on the EEG, which had no behavioral expression. During SE the spike rate was higher in the RLD group than in the SHD group. Overall these results confirm that RLD of KA is a more robust and consistent mouse model of SE than the SHD of KA mouse model.