dictyNews
Electronic Edition
Volume 37, number 6
September 2, 2011
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Abstracts
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Abundant Class of Non-coding RNA Regulates Development in the Social
Amoeba Dictyostelium discoideum
Lotta Avesson1, Heiko T Schumacher2, Pierre Fechter3, Pascale Romby3,
Ulf Hellman4, and Fredrik Söderbom1*
1Department of Molecular Biology, Biomedical Center, Swedish University of
Agricultural Sciences, Box 590, SE-75124 Uppsala, Sweden,
2Present address: Merck Millipore AG, Dammstrasse 19, 6301 Zug, Switzerland,
3Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC,
Strasbourg, France,
4Ludwig Institute for Cancer Research, Ltd. Biomedical Center, Uppsala
University, Box 595 SE-75124 Uppsala, Sweden
RNA Biology, In press
Non-coding (nc)RNAs are important players in most biological processes.
Although small RNAs such as microRNAs and small interfering RNAs have
emerged as exceptionally important regulators of gene expression, great
numbers of larger ncRNAs have also been identified. Many of these are
abundant and differentially expressed but their functions have in most
cases not been elucidated. The social amoeba Dictyostelium discoideum
contain the ncRNAs commonly found in eukaryotes. In addition, we
previously reported the identification of two novel classes of 42-65 nt
long stem-loop forming RNAs, Class I and Class II RNAs, with unknown
function. In this study we have further characterized these abundant
ncRNAs, which are down regulated during development. We have
confirmed expression of 29 Class I RNAs and experimentally verified
the formation of the computationally predicted short conserved stem
structure. Furthermore, we have for the first time created knockout
strains for several small ncRNA genes in D. discoideum and found
that deletion of one of the Class I RNAs, DdR-21, results in aberrant
development. In addition we have shown that this Class I RNA forms
a complex with one or several proteins but do not appear to be
associated with ribosomes or polysomes. In a pull down assay,
several proteins interacting with DdR-21 were identified, one of these
has two RNA recognition motifs (RRMs). The purified RRM containing
protein was demonstrated to bind directly and specifically to DdR-21.
Submitted by: Lotta Avesson <[[log in to unmask]]
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The anti-epileptic drug valproic acid and other medium chain fatty acids
acutely reduce phosphoinositide levels independently of inositol in
Dictyostelium
Pishan Chang1*, Benoit Orabi1*, Rania M. Deranieh2, Manik Dham1,
Oliver Hoeller3, Jakob A Shimshoni4, Boris Yagen4,Meir Bialer4,
Miriam L. Greenberg2, Matthew C. Walker5 and Robin SB Williams1‡
1 Centre for Biomedical Sciences, School of Biological Sciences, Royal
Holloway University of London, Egham, TW20 0EX, UK
2 Department of Biological Sciences, Wayne State University, Detroit, MI
48202, USA
3 Department of Cellular and Molecular Pharmacology and the
Cardiovascular Research Institute, University of California San Francisco,
San Francisco, CA 94158, USA
4 Institute for Drug Research, School of Pharmacy, Faculty of Medicine,
The Hebrew University of Jerusalem, 91120 Jerusalem, Israel
5 Department of Clinical and Experimental Epilepsy, Institute of Neurology,
University College London, London, WC1N 3BG, UK
Disease Models and Mechanisms, in press
Valproic acid (VPA) is the most widely prescribed epilepsy treatment
worldwide, but its mechanism of action remains unclear. Our previous work
identified a previously unknown effect of VPA in reducing phosphoinositide
production in the simple model Dictyostelium followed by the transfer of data
to a mammalian synaptic release model. In our current study, we show that
the reduction in phosphoinositide [PtdInsP (also known as PIP) and PtdInsP2
(also known as PIP2)] production caused by VPA is acute and dose dependent,
and that this effect occurs independently of phosphatidylinositol 3-kinase (PI3K)
activity,inositol recycling and inositol synthesis. In characterising the structural
requirements for this effect, we also identify a family of medium-chain fatty
acids that show increased efficacy compared with VPA. Within the group of
active compounds is a littlestudied group previously associated with seizure
control, and analysis of two of these compounds (nonanoic acid and
4-methyloctanoic acid) shows around a threefold enhanced potency compared
with VPA for protection in an in vitro acute rat seizure model. Together, our
data show that VPA and a newly identified group of medium-chain fatty acids
reduce phosphoinositide levels independently of inositol regulation, and
suggest the reinvestigation of these compounds as treatments for epilepsy.
Submitted by: Williams Robin [[log in to unmask]]
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Conserved Valproic Acid-Induced Lipid Droplet Formation in Dictyostelium
and Human hepatocytes (huh7) Identifies Structurally Active Compounds
Lucy M Elphick1, Nadine Pawolleck1, Irina A Guschina2, Leila Chaieb1,
Daniel Eikel3, Heinz Nau3, John L Harwood2, Nick J Plant4 and
Robin SB Williams1†
1 Centre for Biomedical Sciences, School of Biological Science, Royal
Holloway University of London, Egham, Surrey, TW20 0EX, UK
2 School of Biosciences, Cardiff University, Cardiff CF10 3AX, UK
3 Institute for Food Toxicology and Analytical Chemistry, University of
Veterinary Medicine Hannover, Bischofsholer Damm 15,
30173 Hannover, Germany
4 Centre for Toxicology, Faculty of Health and Medical Sciences,
University of Surrey, Guildford, GU2 7XH, UK
Disease Models and Mechanisms, in press
Lipid droplet formation and subsequent steatosis has been reported to
contribute to hepatotoxicity and is an adverse effect of many pharmacological
agents including the anti-epileptic valproic acid (VPA). In this study, we have
developed a simple model system Dictyostelium discoideum to investigate
the effects of VPA and related compounds in lipid droplet formation. In
mammalian hepatocytes, VPA increases lipid droplet accumulation over
a 24 hour period giving rise to liver cell damage, and we show a similar
effect in Dictyostelium following 30 minute VPA treatment. Using 3H-labelled
poly-unsaturated (arachidonic) or saturated (palmitic) fatty acids, we shown
VPA treatment of Dictyostelium gives rise to an increased accumulation of
both fatty acids in phosphatidylcholine, phosphatidylethanolamine and
non-polar lipids in this time period, with a similar trend observed in Human
hepatocytes (Huh7 cells) labelled with 3H-arachidonic acid. In addition,
pharmacological inhibition of beta-oxidation in Dictyostelium phenocopies
fatty acid accumulation, in agreement with data reported in mammalian
systems. Using Dictyostelium, we then screened a range of VPA-related
compounds to identify compounds with high and low lipid-accumulation
potential, and validated these activities for effects on lipid droplet formation
using human hepatocytes. Structure-activity relationships for these
VPA-related compounds suggest that lipid accumulation is independent
of VPA-catalysed teratogenicity and inositol depletion. These results
suggest that Dictyostelium may provide both a novel model system for
the analysis of lipid droplet formation in human hepatocytes and a rapid
method for identifying VPA-related compounds showing liver toxicology.
Submitted by: Williams Robin [[log in to unmask]]
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Investigating the effect of emetic compounds on chemotaxis in Dictyostelium
identifies a non-sentient model for bitter and hot tastant research
Steven Robery1, Janina Mukanowa1, Nathalie Percie du Sert2,
Paul L. R. Andrews2, Robin SB Williams1,*
1Centre for Biomedical Sciences, School of Biological Sciences, Royal
Holloway University of London, Egham TW20 0EX, UK
2Division of Biomedical Sciences, St George's University of London,
London SW17 0RE, UK
Plos One
Novel chemical entities (NCEs) may be investigated for emetic liability
in a range of unpleasant experiments involving retching, vomiting or
conditioned taste aversion/food avoidance in sentient animals. We have
used a range of compounds with known emetic /aversive properties to
examine the possibility of using the social amoeba, Dictyostelium
discoideum, for research into identifying and understanding emetic
liability, and hence reduce adverse animal experimentation in this area.
Twenty eight emetic or taste aversive compounds were employed to
investigate the acute (10 min) effect of compounds on Dictyostelium
cell behaviour (shape, speed and direction of movement) in a shallow
chemotaxic gradient (Dunn chamber). Compound concentrations were
chosen based on those previously reported to be emetic or aversive in
in vivo studies and results were recorded and quantified by automated
image analysis. Dictyostelium cell motility was rapidly and strongly
inhibited by four structurally distinct tastants (three bitter tasting
compounds - denatonium benzoate, quinine hydrochloride, phenylthiourea,
and the pungent constituent of chilli peppers - capsaicin). In addition,
stomach irritants (copper chloride and copper sulphate), and a
phosphodiesterase IV inhibitor also rapidly blocked movement. A
concentration-dependant relationship was established for five of these
compounds, showing potency of inhibition as capsaicin (IC50=11.9±4.0µM)
> quinine hydrochloride (IC50=44.3±6.8µM) > denatonium benzoate
(IC50=129±4µM) > phenylthiourea (IC50=366±5µM) > copper sulphate
(IC50=1433±3µM). In contrast, 21 compounds within the cytotoxic and
receptor agonist/antagonist classes did not affect cell behaviour. Further
analysis of bitter and pungent compounds showed that the effect on cell
behaviour was reversible and not cytotoxic, suggesting an
uncharacterised molecular mechanism of action for these compounds.
These results therefore demonstrate that Dictyostelium has potential as
a non-sentient model in the analysis of the molecular effects of tastants,
although it has limited utility in identification of emetic agents in general.
Submitted by: Williams Robin [[log in to unmask]]
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[End dictyNews, volume 37, number 6]
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