The use of insect cell line Sf21 for ecotoxicity testing.

Insect cell lines are finding utility in many areas of biology, but their application as an in vitro tool for ecotoxicity testing has been given less attention. Our study aimed to demonstrate the utility and sensitivity of Sf21 cells to commonly used fungicides: Propiconazole and CuSO4, as well as dimethyl sulphoxide (DMSO) an industrial solvent. Sf21 cells were readily cultured from frozen stocks in 3-4 days and showed utility as an invertebrate in vitro acute toxicity test. The data showed the threshold levels of cell survivability against propiconazole and CuSO4. The EC50 values were 135.1 µM and 3.31 mM respectively. The LOAEL (lowest observed adverse effect level) was ≈ 1 µM for propiconazole and ≈ 10 µM for CuSO4. Culturing of Sf21 cells in media containing the solvent DMSO showed that 0.5% DMSO concentration did not effect cell viability. Sf21 cells are sensitive and useful as a robust ecologically relevant screening tool for acute toxicity testing.

A pinch of salt: response of coastal grassland plants to simulated seawater inundation treatments.

BACKGROUND AND AIMS: The combination of rising sea levels and increased storm frequency and intensity is predicted to increase the severity of oceanic storm surge events and the impact of flooding on coastal ecosystems globally. Understanding how plant communities respond to this threat necessitates experiments involving plant immersion in saline water, but logistical issues and natural variation in seawater composition mean that pure NaCl solutions or marine aquarium salts (MS) are widely used. Nonetheless, their comparative impact on plant ecophysiology, and thus relevance to understanding real-world flooding scenarios, is unknown. METHODS: In the first of two experiments, we examined how six ecophysiological responses in white clover (Trifolium repens) varied when plants were subjected to five different inundation treatments: deionized water, natural seawater, an MS solution and two NaCl solutions. In a second experiment, we examined how immersion in deionized water, MS solution and natural seawater affected six European perennial herb species, three native to Spanish sand dunes, and three from British coastal grasslands. RESULTS: The two NaCl solutions induced exceptional Trifolium mortality, but responses varied little between MS and seawater treatments. In the second experiment, although leaf tissue necrosis and proline concentrations increased, and growth decreased compared with untreated controls, only one response in one species varied between MS and seawater treatments. Chemical speciation modelling revealed major variation in free Na+ and Cl- between NaCl solutions and seawater, but minor differences between MS and seawater. CONCLUSIONS: We show that NaCl solutions are unsuitable surrogates to investigate plant response to elevated environmental salinity. Although responses to natural seawater and MS were consistent within species, there was notable between-species variation. Consequently, the first steps to elucidating how these species-specific responses influence coastal plant community recovery following storm surge can likely be achieved using commercial marine aquarium salts as substitutes for natural seawater.

Physiological niche and geographical range in European diving beetles (Coleoptera: Dytiscidae).

Geographical ranges vary greatly in size and position, even within recent clades, but the factors driving this remain poorly understood. In aquatic beetles, thermal niche has been shown to be related to both the relative range size and position of congeners but whether other physiological parameters play a role is unknown. Metabolic plasticity may be critical for species occupying more variable thermal environments and maintaining this plasticity may trade-off against other physiological processes such as immunocompetence. Here we combine data on thermal physiology with measures of metabolic plasticity and immunocompetence to explore these relationships in Deronectes (Dytiscidae). While variation in latitudinal range extent and position was explained in part by thermal physiology, aspects of metabolic plasticity and immunocompetence also appeared important. Northerly distributed, wide-ranging species apparently used different energy reserves under thermal stress from southern endemic congeners and differed in their antibacterial defences. This is the first indication that these processes may be related to geographical range, and suggests parameters that may be worthy of exploration in other taxa.

Large changes in NAD levels associated with CD38 expression during HL-60 cell differentiation.

NAD is an important cofactor involved in multiple metabolic reactions and as a substrate for several NAD-dependent signalling enzymes. One such enzyme is CD38 which, alongside synthesising Ca(2+)-releasing second messengers and acting as a cell surface receptor, has also been suggested to play a key role in NAD(+) homeostasis. CD38 is well known as a negative prognostic marker in B-CLL but the role of its enzymatic activity has not been studied in depth to date. We have exploited the HL-60 cell line as a model of inducible CD38 expression, to investigate CD38-mediated regulation intracellular NAD(+) levels and the consequences of changes in NAD(+) levels on cell physiology. Intracellular NAD(+) levels fell with increasing CD38 expression and this was reversed with the CD38 inhibitor, kuromanin confirming the key role of CD38 in NAD(+) homeostasis. We also measured the consequences of CD38 expression during the differentiation on a number of functions linked to NAD(+) and we show that some but not all NAD(+)-dependent processes are significantly affected by the lowered NAD(+) levels. These data suggest that both functional roles of CD38 might be important in the pathogenesis of B-CLL.

Galleria mellonella larvae exhibit a weight-dependent lethal median dose when infected with methicillin-resistant Staphylococcus aureus.

Galleria mellonella is a recognised model to study antimicrobial efficacy; however, standardisation across the scientific field and investigations of methodological components are needed. Here, we investigate the impact of weight on mortality following infection with Methicillin-resistant Staphylococcus aureus (MRSA). Larvae were separated into six weight groups (180-300 mg at 20 mg intervals) and infected with a range of doses of MRSA to determine the 50% lethal dose (LD50), and the 'lipid weight' of larvae post-infection was quantified. A model of LD50 values correlated with weight was developed. The LD50 values, as estimated by our model, were further tested in vivo to prove our model. We establish a weight-dependent LD50 in larvae against MRSA and demonstrate that G. mellonella is a stable model within 180-260 mg. We present multiple linear models correlating weight with: LD50, lipid weight, and larval length. We demonstrate that the lipid weight is reduced as a result of MRSA infection, identifying a potentially new measure in which to understand the immune response. Finally, we demonstrate that larval length can be a reasonable proxy for weight. Refining the methodologies in which to handle and design experiments involving G. mellonella, we can improve the reliability of this powerful model.

NAADP-induced Ca(2+ signaling in response to endothelin is via the receptor subtype B and requires the integrity of lipid rafts/caveolae.

We have investigated the role of NAADP-mediated Ca(2+) mobilization in endothelin (ET) signaling via endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) in rat peritubular smooth muscle cells. Microinjection and extracellular application of NAADP were both able to elicit Ca(2+) release which was blocked by inhibitory concentrations of NAADP, by impairing Ca(2+) uptake in acidic stores with bafilomycin, and by thapsigargin. Ca(2+) release in response to selective ETB stimulation was abolished by inhibition of NAADP signaling through the same strategies, while these treatments only partially impaired ETA-dependent Ca(2+) signaling, showing that transduction of the ETB signal is dependent on NAADP. In addition, we show that lipid rafts/caveolae contain ETA, ETB, and NAADP/cADPR generating enzyme CD38 and that stimulation of ETB receptors results in increased CD38 activity; interestingly, ETB- (but not ETA-) mediated Ca(2+) responses were antagonized by disruption of lipid rafts/caveolae with methyl-beta-cyclodextrin. These data demonstrate a primary role of NAADP in ETB-mediated Ca(2+) signaling and strongly suggest a novel role of lipid rafts/caveolae in triggering ET-induced NAADP signaling.

PPADS is a reversible competitive antagonist of the NAADP receptor.

NAADP has been shown to act as a second messenger in a wide range of systems from plants to mammalian cells. Although it had always been considered as a canonical second messenger, recent work has shown that it is also active when applied extracellularly. It has also been suggested that NAADP might have a direct action on P2 receptors, based on the action of a pharmacological agent, PPADS, on Ca2+ signals in response to extracellular NAADP. We have therefore investigated whether PPADS can act directly on the intracellular NAADP-induced Ca2+-release system in the well characterised sea urchin egg homogenate system. Indeed, PPADS, and its structural analogue PPNDS were able to compete with [32P]NAADP for the binding site and binding curves revealed that both compounds display affinities in the low micromolar range. The binding of PPADS was reversible in contrast to that of NAADP. In fluorimetric Ca2+-release experiments, PPADS was able to competitively antagonise NAADP-induced Ca2+-release with an IC50 of 20 microM, while it did not affect the other Ca2+-release channels. This is the first report of a reversible, competitive antagonist of the sea urchin NAADP receptor. Furthermore, PPADS might reveal itself as an invaluable tool to investigate NAADP signalling and is a lead compound for the synthesis of potent and specific antagonists.

NAADP: an atypical Ca2+-release messenger?

Recently, nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown, using different techniques, to mobilize intracellular Ca2+ stores in invertebrate, lower vertebrate, plant and mammalian cells. This endogenous molecule might play an atypical role in Ca2+ signalling by coordinating the responses of other Ca2+-releasing messengers. Furthermore, radioligand binding experiments have provided an insight into how desensitization of the NAADP receptor might occur.

Production and characterization of reduced NAADP (nicotinic acid-adenine dinucleotide phosphate).

The pyridine nucleotide NAADP (nicotinic acid-adenine dinucleotide phosphate) has been shown to act as a Ca2+-releasing intracellular messenger in a wide variety of systems from invertebrates to mammals and has been implicated in a number of cellular processes. NAADP is structurally very similar to its precursor, the endogenous coenzyme NADP and while much is known about the reduced form of NADP, NADPH, it is not known whether NAADP can also exist in a reduced state. Here we report that NAADP can be reduced to NAADPH by endogenous cellular enzymes and that NAADPH is functionally inert at the NAADP receptor. These data suggest that NAADPH could represent a mechanism for rapidly inactivating NAADP in cells.

Triazine dyes are agonists of the NAADP receptor.

NAADP has been shown to be a potent calcium-releasing second messenger in a wide variety of cell types to date. However, research has been hampered by a lack of pharmacological agents, with which to investigate NAADP-induced calcium release, and by the molecular identity of its cellular target protein being unknown. In the present paper, the sea urchin egg model was used to investigate whether triazine dyes, which can act as nucleotide mimetics, can bind to the NAADP receptor, induce Ca(2+) release and be used for affinity chromatography of the receptor. Indeed, all the triazine dyes tested (Reactive Red 120 (RR120), Reactive Green 19 (RG19), Reactive Green 5 (RG5), Cibacron Blue 3GA and Reactive Yellow 86) displayed micromolar affinities, except for Reactive Orange 14. Furthermore, unlike NAADP, RR120, RG19 and RG5 did not bind in an irreversible manner. The compound that displayed the highest affinity, RR120, was tested in a (45)Ca(2+) efflux assay. This compound released Ca(2+) via the NAADP receptor, as shown by the ability of subthreshold NAADP concentrations to inhibit this release. Furthermore, heparin and ruthenium red were unable to block RR120-induced Ca(2+) release. We have also shown that RG5 and RG19, immobilised on resins, retain the ability to bind to the receptor, and that this interaction can be disrupted by high salt concentrations. As a proof of principle, we have shown that this can be used to partially purify the NAADP receptor by at least 75-fold. In conclusion, triazine dyes interact with the NAADP receptor, and this could be exploited in future to create a new generation of pharmacological tools to investigate this messenger and, in combination with other techniques, to purify the receptor.

Wld S protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice.

The slow Wallerian degeneration (Wld(S)) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70-amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide-synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of Wld(S)-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the Wld(S) VCP-binding domain with an alternative ataxin-3-derived VCP-binding sequence restores its protective function. Enzyme-dead Wld(S) is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. Wld(S) requires both of its components to protect axons from degeneration.

NAD depletion by FK866 induces autophagy.

NAD is a multifunctional molecule involved in both metabolic processes and signaling pathways. Such signalling pathways consume NAD which is replenished via one of several biosynthesis pathways. We show that influx of NAD across the plasma membrane may be able to contribute to the homeostasis of intracellular NAD levels. Indeed, extracellular application of NAD was able to replete NAD levels that had been lowered pharmacologically using the novel drug FK866 and was also able to rescue cells from FK866-induced cell death. A marked lag between the drop in NAD levels and cell death prompted us to investigate the mechanism of cell death. We were unable to find evidence of apoptosis as assessed by immunoblotting for the Caspase 3 activation fragment and immunostaining for cytochrome C and AIF translocation. We, therefore, investigated whether autophagy was initiated by FK866. Indeed, we were able to observe the formation of LC3-positive vesicles that had fused with lysosomes in FK866-treated but not control cells. Furthermore, this autophagic phenotype could be reverted by the addition of NAD to the extracellular medium.

Click chemistry reactions in medicinal chemistry: applications of the 1,3-dipolar cycloaddition between azides and alkynes.

In recent years, there has been an ever-increasing need for rapid reactions that meet the three main criteria of an ideal synthesis: efficiency, versatility, and selectivity. Such reactions would allow medicinal chemistry to keep pace with the multitude of information derived from modern biological screening techniques. The present review describes one of these reactions, the 1,3-dipolar cycloaddition ("click-reaction") between azides and alkynes catalyzed by copper (I) salts. The simplicity of this reaction and the ease of purification of the resulting products have opened new opportunities in generating vast arrays of compounds with biological potential. The present review will outline the accomplishments of this strategy achieved so far and outline some of medicinal chemistry applications in which click-chemistry might be relevant in the future.

Characterization of NAD uptake in mammalian cells.

Recent evidence has shown that NAD(P) plays a variety of roles in cell-signaling processes. Surprisingly, the presence of NAD(P) utilizing ectoenzymes suggests that NAD(P) is present extracellularly. Indeed, nanomolar concentrations of NAD have been found in plasma and other body fluids. Although very high concentrations of NAD have been shown to enter cells, it is not known whether lower, more physiological concentrations are able to be taken up. Here we show that two mammalian cell types are able to transport low NAD concentrations effectively. Furthermore, extracellular application of NAD was able to counteract FK866-induced cell death and restore intracellular NAD(P) levels. We propose that NAD uptake may play a role in physiological NAD homeostasis.

Synthesis and biological evaluation of isosteric analogues of FK866, an inhibitor of NAD salvage.

One of the great challenges of medicinal chemistry is to create novel, effective, chemotherapeutic agents that show specificity for cancer cells combined with low systemic toxicity. A novel idea is to target the enzymes of the NAD biosynthesis and recycling pathways given that cancer cells display a higher NAD turnover rate than healthy cells. To this end, the compound FK866 (APO866; (E)-N-[4-(1-benzoylpiperidin-4-yl) butyl]-3-(pyridin-3-yl) acrylamide), which blocks nicotinamide phosphoribosyltransferase (NMPRTase) has entered clinical trials as a potential chemotherapeutic agent. Here we report the synthesis of analogues of FK866 synthesized by click chemistry.

Emerging functions of extracellular pyridine nucleotides.

In addition to the well-known metabolic functions of NAD and NADP, it is rapidly emerging that these 2 pyridine nucleotides and their derivatives also play important roles in cell signaling. Surprisingly, a number of NAD(P) metabolizing enzymes and NAD(P) targets have been found on the outer surface of the plasma membrane and the presence of NAD has been confirmed in extracellular fluids. These findings have opened the door to a new field of research aimed at elucidating the contribution of extracellular pyridine nucleotides in physiological signaling pathways and pathological conditions.

Characterization of cyclic adenine dinucleotide phosphate ribose levels in human spermatozoa.

OBJECTIVE: To determine the presence of the Ca2+-releasing pyridine nucleotide derivative, cyclic adenine dinucleotide phosphate ribose (cADPR), in human spermatozoa and to investigate its role in progesterone-induced Ca2+ oscillations in spermatozoa. DESIGN: Biochemical investigation on human spermatozoa from healthy volunteers. SETTING: Healthy volunteers in an academic research environment. PATIENT(S): Ten volunteers. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The cADPR levels. RESULT(S): Human spermatozoa contain micromolar concentrations of cADPR that do not change significantly during sperm capacitation. An active synthetic machinery for cADPR is present in human spermatozoa, whereas degradation activity is minimal. Although progesterone-induced Ca2+ oscillations are dependent on the ryanodine receptor, they are unaffected by cADPR antagonists. CONCLUSION(S): It appears that cADPR does not to play a role in Ca2+ oscillations in spermatozoa, but the presence of high concentrations of cADPR suggests that, instead, it may be introduced into the egg at fertilization and play a role in the Ca2+ transient immediately following sperm-egg fusion.

SERCA-inhibiting activity of C-19 terpenolides from Thapsia garganica and their possible biogenesis.

An investigation of Thapsia garganica afforded a series of tetracyclic C-19 dilactones, whose production was dependent on the time and location of the collection. These unusual tetrahomosesquiterpenoids are presumably biosynthesized via a carbon dioxide-triggered electrophilic polyolefin cyclization. Despite the structural differences with thapsigargin, these compounds showed SERCA-inhibiting properties.

Effect of luminal and extravesicular Ca2+ on NAADP binding and release properties.

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to be a powerful Ca2+ release agent in numerous systems, including echinoderms, plants, and mammalian cells. NAADP has been shown to release Ca2+ via a separate mechanism to IP3 and ryanodine receptors, and specific binding sites have recently been characterised. However, functional studies have shown that there is a functional interplay between the NAADP-sensitive mechanism and the other two. In particular, it appears that activation of the NAADP receptor might act as a trigger to facilitate responses from IP3 and ryanodine receptors. To further characterise this interplay, we have investigated the effects of luminal and cytosolic Ca2+ on the NAADP receptor in sea urchin egg homogenates. We report that neither cytosolic nor luminal Ca2+ appears to influence NAADP binding. Conversely, emptying of stores significantly amplifies NAADP-induced fractional Ca2+-release, providing a mechanism of self-adjustment independent of store loading.