Discovery of novel sodium channel inhibitors—A gene family-based approach

Voltage-gated sodium (Na_V) channel inhibitors are an important class of drugs that are used to treat a number of CNS indications including pain, local anaesthesia, epilepsy and bipolar disorder. These drugs all have their origins in traditional “empirical” pharmacology, and it was only some time after discovery that they were found to inhibit Na_V channels. The basis for therapeutic selectivity of these drugs within different disease indications is currently unknown. However, the subsequent discovery of a multi-gene family of Na_V channels suggests a possible mechanism and has opened the way for more targeted approaches to finding improved therapeutic inhibitors. This article describes some ongoing approaches to systematically clone, express and characterise the entire family of Na_V subtypes in order to better understand their properties and define their individual physiological and pathophysiological roles. As well as providing specific disease validation for individual subtypes, this also provides a panel of reagents for comprehensively exploring the efficacy, selectivity and potency relationships of existing Na_V-blocking drugs. In this way, a gene family-based approach to Na_V channels has enabled a “drug-to-target” approach, reversing the more usual “gene-to-target-to-drug” paradigm. Together with recent advances in assay technology, gene family-based approaches are increasing the tractability of these targets and are re-invigorating Na_V drug discovery within the pharmaceutical industry.     

Acid phosphatase activity in intercellular spaces in roots of Nymphoides peltata (S. G. Gmel.) O. Kuntze

Abstract. Acid phosphatase was found cytochemically in intercellular spaces in the root of Nymphoides peltata. Different methods, using lead salts and azo-dyes, gave similar results. Reaction product appeared on material, possibly cytoplasmic, within the intercellular spaces and also against the outer walls of cells which formed the intercellular spaces. Possible functions of acid phosphatase in intercellular spaces are discussed.     

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.     

Lead increases urinary zinc excretion in rats

The major purpose of this study was to determine whether acute or chronic Pb exposure would increase urinary excretion of zinc in the rat. Four groups of unanesthetized rats were given 0, 0.03, 0.3, or 3 mg Pb (as acetate) kg intravenously, and urinary excretion of zinc, sodium, and potassium was monitored for 6 h. Only at the highest dose was urinary Zn excretion significantly elevated; there were no significant changes in sodium and potassium excretion at any dose. Two other groups of rats were studied for 9 weeks in metabolism cages before and during administration of either 500 ppm Pb (as acetate) or equimolar Na acetate in the drinking water. Two days after Pb treatment and continuing through day 35, Zn excretion was elevated in the Pb-exposed animals; beyond this day, zinc excretion became similar in the two groups. The difference in Zn excretion was not the result of lower water intake by the Pb-treated animals. At sacrifice (70 days after starting Pb exposure), Pb-exposed animals had lower Zn content of the plasma and testis, but there was no difference in kidney Zn. Plasma renin activity was significantly higher in Pb-exposed animals. We conclude that chronic Pb exposure in rats can result in some degree of decreased tissue zinc, which is, at least in part, secondary to increased urinary losses of zinc.     

Biological monitoring of exposure to ambient lead and cadmium using avian feathers: A study from Northern India

Biological monitoring of exposure to ambient environmental lead and cadmium was performed using feathers of 26 species of birds native to Western Uttar Pradesh in Northern India. The rationale of this study was to address three questions. First, is there any avian species that can be treated as a suitable bioindicator of lead or cadmium present in the environment? Second, do the birds selectively accumulate lead and cadmium in their feathers and exhibit interspecies variation? Third, is there any threat to endangered species of this region from metal pollution? Average concentration of lead in the feathers of selected birds ranged from 3.40 µg/g in parrot to 301.6 µg/g in golden pheasant, whereas cadmium concentration was higher ranging from 40.20 µg/g in red crow to 450 µg/g in blue macaw. A comparison of results made through ANOVA revealed a significant difference in the concentration of lead (df = 25; F = 3965.54) and cadmium (df = 25; F = 8537.27) in their feathers. We hypothesize that feathers of synanthropic birds may be treated as suitable noninvasive tool to monitor the ambient environmental contamination by lead and cadmium. Their accumulation in endangered birds may lead to population decline causing serious ecological disturbances in the region.     

Heavy metals in aquatic macrophytes drifting in a large river

Macrophytes drifting throughout the water column in the Detroit River were collected monthly from May to October 1985 to estimate the quantities of heavy metals being transported to Lake Erie by the plants. Most macrophytes (80–92% by weight) drifted at the water surface. Live submersed macrophytes made up the bulk of each sample. The most widely distributed submersed macrophyte in the river, American wildcelery (Vallisneria americana), occurred most frequently in the drift. A total of 151 tonnes (ash-free dry weight) of macrophytes drifted out of the Detroit River from May to October. The drift was greatest (37 tonnes) in May. Concentrations of heavy metals were significantly higher in macrophytes drifting in the river than in those growing elsewhere in unpolluted waters. Annually, a maximum of 2 796 kg (eight heavy metals combined) were transported into Lake Erie by drifting macrophytes. The enrichment of all metals was remarkably high (range: 4 000 × to 161 000 × ) in macrophytes, relative to their concentration in water of the Detroit River. Detroit River macrophytes are thus a source of contaminated food for animals in the river and in Lake Erie.Contribution 734 of the National Fisheries Research Center-Great Lakes, U.S. Fish and Wildlife Service, 1451 Green Road, Ann Arbor, MI 48105.     

Application of Poly 1,8‐diaminonaphthalene/multiwalled carbon nanotubes‐COOH hybrid material as an efficient sorbent for trace determination of cadmium and lead ions in water samples

Poly 1,8‐diaminonaphthalene/multiwalled carbon nanotubes‐COOH hybrid material as an effective sorbents in solid phase extraction has been developed for the separation and preconcentration of Cd(II) and Pb(II) at trace levels in environmental water samples. The results indicate that the novel nanocomposite show a high affinity for these heavy metals due to the presence of several good extractive sites, which are introduced to the synthesized nanocomposite The maximum adsorption capacity of the synthesized sorbent for cadmium and lead ions was found to be 101.2 and 175.2 mg g^?1, respectively. The detection limits of this method were 0.09 and 0.7 ng ml^?1 for Cd(II) and Pb(II), respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Plant cell culture technology–harnessing a biological approach for competitive cyclotides production

Cyclotides are naturally occurring mini-proteins that have a cyclic backbone and a knotted arrangement of three disulfide bonds. They are remarkably stable and have a diverse range of therapeutically useful biological activities, including antimicrobial and anti-HIV activity, although their natural function appears to be plant defence agents. Cyclotides are amenable to chemical synthesis; however currently most bioactivity studies have involved the use of peptides extracted from plants. Plant cell culture technology shows promise towards the goal of producing therapeutically active cyclotides in qualities and quantities required for drug development.     

A new target for Parkinson’s disease: Small molecule SERCA activator CDN1163 ameliorates dyskinesia in 6-OHDA-lesioned rats

Endoplasmic reticulum (ER) stress is intimately linked to Parkinson’s disease (PD) pathophysiology. Disrupted intracellular calcium homeostasis is a major cause of the ER stress seen in dopaminergic neurons, leading to the cell death and subsequent loss of movement and coordination in patients. Dysfunctional calcium handling proteins play a major role in the promulgation of ER stress in PD. Specifically, compromised sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) has been identified as a major cause of ER stress and neuron loss in PD. We have identified a small molecule activator of SERCA that increases ER calcium content, rescues neurons from ER stress-induced cell death in vitro, and shows significant efficacy in the rat 6-hydroxydopamine (6-OHDA) model of PD. Together, these results support targeting SERCA activation as a viable strategy to develop disease-modifying therapeutics for PD.