61 research outputs found
A Small Conductance Calcium-Activated K<sup>+</sup> Channel in C. elegans, KCNL-2, Plays a Role in the Regulation of the Rate of Egg-Laying
In the nervous system of mice, small conductance calcium-activated potassium (SK) channels function to regulate neuronal excitability through the generation of a component of the medium afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson's disease. Due to the complexity of studying protein function in the mammalian nervous system, we sought to characterize an SK channel homologue, KCNL-2, in C. elegans, a genetically tractable system in which the lineage of individual neurons was mapped from their early developmental stages. Sequence analysis of the KCNL-2 protein reveals that the six transmembrane domains, the potassium-selective pore and the calmodulin binding domain are highly conserved with the mammalian homologues. We used widefield and confocal fluorescent imaging to show that a fusion construct of KCNL-2 with GFP in transgenic lines is expressed in the nervous system of C. elegans. We also show that a KCNL-2 null strain, kcnl-2(tm1885), demonstrates a mild egg-laying defective phenotype, a phenotype that is rescued in a KCNL-2-dependent manner. Conversely, we show that transgenic lines that overexpress KCNL-2 demonstrate a hyperactive egg-laying phenotype. In this study, we show that the vulva of transgenic hermaphrodites is highly innervated by neuronal processes and by the VC4 and VC5 neurons that express GFP-tagged KCNL-2. We propose that KCNL-2 functions in the nervous system of C. elegans to regulate the rate of egg-laying. © 2013 Chotoo et al
Cannabinoid Agonists Inhibit Neuropathic Pain Induced by Brachial Plexus Avulsion in Mice by Affecting Glial Cells and MAP Kinases
Many studies have shown the antinociceptive effects of cannabinoid (CB) agonists in different models of pain. Herein, we have investigated their relevance in neuropathic pain induced by brachial plexus avulsion (BPA) in mice.Mice underwent BPA or sham surgery. The mRNA levels and protein expression of CB(1) and CB(2) receptors were assessed by RT-PCR and immunohistochemistry, respectively. The activation of glial cells, MAP kinases and transcription factors were evaluated by immunohistochemistry. The antinociceptive properties induced by cannabinoid agonists were assessed on the 5(th) and 30(th) days after surgery. We observed a marked increase in CB(1) and CB(2) receptor mRNA and protein expression in the spinal cord and dorsal root ganglion, either at the 5(th) or 30(th) day after surgery. BPA also induced a marked activation of p38 and JNK MAP kinases (on the 30(th) day), glial cells, such as microglia and astrocytes, and the transcription factors CREB and NF-κB (at the 5(th) and 30(th) days) in the spinal cord. Systemic treatment with cannabinoid agonists reduced mechanical allodynia on both the 5(th) and 30(th) days after surgery, but the greatest results were observed by using central routes of administration, especially at the 30(th) day. Treatment with WIN 55,212-2 prevented the activation of both glial cells and MAP kinases, associated with an enhancement of CREB and NF-κB activation.Our results indicate a relevant role for cannabinoid agonists in BPA, reinforcing their potential therapeutic relevance for the management of chronic pain states
In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus: a biotechnological approach to study pine wilt disease
Abstract
Main conclusion Co-cultures of Pinus pinaster with
Bursaphelenchus xylophilus were established as a
biotechnological tool to evaluate the effect of nematotoxics
addition in a host/parasite culture system.
The pinewood nematode (PWN), Bursaphelenchus xylophilus,
the causal agent of pine wilt disease (PWD), was
detected for the first time in Europe in 1999 spreading
throughout the pine forests in Portugal and recently in
Spain. Plant in vitro cultures may be a useful experimental
system to investigate the plant/nematode relationships in
loco, thus avoiding the difficulties of field assays. In this
study, Pinus pinaster in vitro cultures were established and
compared to in vivo 1 year-old plantlets by analyzing shoot
structure and volatiles production. In vitro co-cultures were
established with the PWN and the effect of the phytoparasite
on in vitro shoot structure, water content and
volatiles production was evaluated. In vitro shoots showed
similar structure and volatiles production to in vivo maritime
pine plantlets. The first macroscopic symptoms of
PWD were observed about 4 weeks after in vitro co-culture
establishment. Nematode population in the culture medium
increased and PWNs were detected in gaps of the callus
tissue and in cavities developed from the degradation of
cambial cells. In terms of volatiles main components,
plantlets, P. pinaster cultures, and P. pinaster with B. xylophilus
co-cultures were all b- and a-pinene rich. Cocultures
may be an easy-to-handle biotechnological approach
to study this pathology, envisioning the understanding
of and finding ways to restrain this highly
devastating nematode.
Keywords Maritime pine ! Monoxenic culture !
Pinewood nematode ! Relative water content ! Shoots
structure ! Volatiles
Abbreviations
BAP 6-Benzylaminopurine
DAI Days after inoculation
EPPO European and Mediterranean Plant Protectio
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