In vitro neuropharmacological evaluation of penitrem-induced tremorgenic syndromes: importance of the GABAergic system

The effects of the fungal neurotoxin penitrem A on the GABAergic and glutamatergic systems in rat brain were evaluated. Penitrem A inhibited binding of the GABA_A-receptor ligand [³H]TBOB to rat forebrain and cerebellar membrane preparations with IC₅₀ (half maximal inhibitory concentration) values of 11 and 9 μM, respectively. Furthermore, penitrem A caused a concentration-dependent increase of [³H]flunitrazepam and [³H]muscimol binding in rat forebrain, but not in cerebellar preparations. The stimulation of [³H]flunitrazepam binding by penitrem A was abolished by the addition of GABA. In cerebellar preparations, a different pharmacological profile was found, with penitrem A allosterically inhibiting [³H]TBOB binding by interacting with a bicuculline-sensitive site. Moreover, penitrem A inhibited the high affinity uptake of GABA and glutamate into cerebellar synaptosomes with IC₅₀ values of 20 and 47 μM, respectively. The toxin showed no effect on NMDA or AMPA glutamate receptor binding. In conclusion, our results suggest that penitrem A exerts region-specific effects in the brain, leading to positive modulation of GABA_A-receptor function in forebrain. Conversely, penitrem A may act as a bicuculline-like convulsant in cerebellum.     

Tales from an academic RNAi screening facility; FAQs

RNAi technology is now a well-established and widely employed research technique that has been adopted by many researchers for use in large-scale screening campaigns. Here, we offer our experience of genome-wide siRNA screening from the perspective of a facility providing screening as a service to a wide range of researchers with diverse interests and approaches. We have experienced the emotional rollercoaster of screening from the exuberant early promise of a screen, the messy reality of the data through to the recognition of screen data as a potential information goldmine. Here, we use some of the questions we most frequently encounter to highlight the initial concerns of many researchers embarking on a siRNA screen and conclude that an informed view of what can be reasonably expected from a screen is essential to the most effective implementation of the technology. Along the way, we suggest that for this area of research at least, either centralization of the resources or close and open collaboration between interested parties offers distinct advantages.     

Genetic differentiation among sympatric cuckoo host races: males matter

Generalist parasites regularly evolve host-specific races that each specialize on one particular host species. Many host-specific races originate from geographically structured populations where local adaptations to different host species drive the differentiation of distinct races. However, in sympatric populations where several host races coexist, gene flow could potentially disrupt such host-specific adaptations. Here, we analyse genetic differentiation among three sympatrically breeding host races of the brood-parasitic common cuckoo, Cuculus canorus. In this species, host-specific adaptations are assumed to be controlled by females only, possibly via the female-specific W-chromosome, thereby avoiding that gene flow via males disrupts local adaptations. Although males were more likely to have offspring in two different host species (43% versus 7%), they did not have significantly more descendants being raised outside their putative foster species than females (9% versus 2%). We found significant genetic differentiation for both biparentally inherited microsatellite DNA markers and maternally inherited mitochondrial DNA markers. To our knowledge, this is the first study that finds significant genetic differentiation in biparentally inherited markers among cuckoo host-specific races. Our results imply that males also may contribute to the evolution and maintenance of the different races, and hence that the genes responsible for egg phenotype may be found on autosomal chromosomes rather than the female-specific W-chromosome as previously assumed.     

Increasing evidence of the role of gene flow in animal evolution: hybrid speciation in the yellow-rumped warbler complex

In this issue of Molecular Ecology, Brelsford et al. (2011) present strong evidence for a case of hybrid speciation within the yellow-rumped warbler complex. Although homoploid hybrid speciation has now been documented in many animals (Mallet 2007), it seems rare in tetrapods (Mavárez & Linares 2008) and it has barely even been mentioned in birds (Price 2008). Brelsford and colleagues thus present the first detailed molecular evidence suggesting that hybrid speciation can occur in birds. Brelsford et al. (2011) posit that Audubon's warbler (Dendroica auduboni) constitutes a hybrid species originating from the admixture of two distinct parental lineages, represented today by myrtle warbler (D. coronata) and black-fronted warbler (D. nigrifrons). The authors present three major lines of molecular evidence suggesting that this is not simply a case of a hybrid swarm or limited introgression.     

IsobariQ: software for isobaric quantitative proteomics using IPTL, iTRAQ, and TMT

Isobaric peptide labeling plays an important role in relative quantitative comparisons of proteomes. Isobaric labeling techniques utilize MS/MS spectra for relative quantification, which can be either based on the relative intensities of reporter ions in the low mass region (iTRAQ and TMT) or on the relative intensities of quantification signatures throughout the spectrum due to isobaric peptide termini labeling (IPTL). Due to the increased quantitative information found in MS/MS fragment spectra generated by the recently developed IPTL approach, new software was required to extract the quantitative information. IsobariQ was specifically developed for this purpose; however, support for the reporter ion techniques iTRAQ and TMT is also included. In addition, to address recently emphasized issues about heterogeneity of variance in proteomics data sets, IsobariQ employs the statistical software package R and variance stabilizing normalization (VSN) algorithms available therein. Finally, the functionality of IsobariQ is validated with data sets of experiments using 6-plex TMT and IPTL. Notably, protein substrates resulting from cleavage by proteases can be identified as shown for caspase targets in apoptosis.     

Identification of the Hydroxyl Radical and Other Reactive Oxygen Species in Human Neutrophil Granulocytes Exposed to a Fragment of the Amyloid Beta Peptide

A fragment of the amyloid beta protein, &#103 A(25-35), was investigated for its effect on production of reactive oxygen species (ROS) in human neutrophil granulocytes. The formation and identification of ROS were examined by using a 2',7'-dichlorofluorescin (DCF) fluorescence assay, a luminol chemiluminescence assay, electron paramagnetic resonance (EPR) spectroscopy with DEPMPO as a spin trap, and hydroxylation of 4-hydroxybenzoate (4-HBA). The DCF assay showed that &#103 A(25-35) stimulated formation of ROS in a concentration and time dependent manner. The inverted peptide, &#103 A(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by &#103 A(25-35). Incubation with diethyldithiocarbamate (a superoxide dimustase inhibitor) and salicylhydroxamate (SHA; a myeloperoxidase inhibitor) reduced the chemiluminescence. This indicates that hypochlorous acid (HOCl) is formed after exposure to &#103 A(25-35). The EPR spectra indicated a concentration dependent formation of superoxide ( O 2 &#148 &#109 ) - and hydroxyl ( &#148 OH)- radicals. Hydroxylation of 4-HBA to 3,4,-dihydroxybenzoate confirmed production of &#148 OH. This response was attenuated by SHA, indicating involvement of HOCl in formation of &#148 OH. The DCF fluorescence was inhibited with U0126 (an extracellular signal regulated protein kinase (ERK) inhibitor). Further analysis with western blot confirmed phosphorylation of ERK1/2 after exposure to &#103 A(25-35). The phospholipase A 2 (PLA 2 ) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, and diphenyleneiodonium, which inhibits the NADPH oxidase, also led to a reduction of the DCF fluorescence. The present findings indicate that &#103 A(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA 2 . Production of O 2 &#148 &#109 can lead to HOCl and further formation of &#148 OH, which both have a cytotoxic potential.     

Amino acid neurotransmission: Dynamics of vesicular uptake

Glutamate, GABA and glycine, the major neurotransmitters in CNS, are taken up and stored in synaptic vesicles by a Mg²⁺-ATP dependent process. The main driving force for vesicular glutamate uptake is the membrane potential, whereas both the membrane potential and the proton gradient contribute to the uptake of GABA and glycine. Glutamate is taken up by a specific transporter with no affinity for aspartate. Evans blue and related dyes are competitive inhibitors of the uptake of glutamate. GABA, β-alanine, and glycine are taken up by the same family of transporter molecules. Aspartate, taurine, and proline are not taken up by any synaptic vesicle preparations. It is suggested that vesicular uptake and release are characteristics that identify these amino acids as neurotransmitters. We also discuss that “quanta” in the brain are not necessarily related the content of neurotransmitter in the synaptic vesicles, but rather to postsynaptic events. Special issue dedicated to Dr. Herman Bachelard.     

Leaf Litter Inhibits Growth of an Amphibian Fungal Pathogen

Past studies have found a heterogeneous distribution of the amphibian chytrid fungal pathogen, Batrachochytrium dendrobatidis (Bd). Recent studies have accounted for some of this heterogeneity through a positive association between canopy cover and Bd abundance, which is attributed to the cooling effect of canopy cover. We questioned whether leaf litter inputs that are also associated with canopy cover might also alter Bd growth. Leaf litter inputs exhibit tremendous interspecific chemical variation, and we hypothesized that Bd growth varies with leachate chemistry. We also hypothesized that Bd uses leaf litter as a growth substrate. To test these hypotheses, we conducted laboratory trials in which we exposed cultures of Bd to leachate of 12 temperate leaf litter species at varying dilutions. Using a subset of those 12 litter species, we also exposed Bd to pre-leached litter substrate. We found that exposure to litter leachate and substrate reduced Bd spore and sporangia densities, although there was substantial variation among treatments. In particular, Bd densities were inversely correlated with concentrations of phenolic acids. We conducted a field survey of phenolic concentrations in natural wetlands which verified that the leachate concentrations in our lab study are ecologically relevant. Our study reinforces prior indications that positive associations between canopy cover and Bd abundance are likely mediated by water temperature effects, but this phenomenon might be counteracted by changes in aquatic chemistry from leaf litter inputs.     

Disappearance of eggs from nonparasitized nests of brood parasite hosts: the evolutionary equilibrium hypothesis revisited

The evolutionary equilibrium hypothesis was proposed to explain variation in egg rejection rates among individual hosts (intra‐ and interspecific) of avian brood parasites. Hosts may sometimes mistakenly reject own eggs when they are not parasitized (i.e. make recognition errors). Such errors would incur fitness costs and could counter the evolution of host defences driven by costs of parasitism (i.e. creating equilibrium between acceptors and rejecters within particular host populations). In the present study, we report the disappearance of host eggs from nonparasitized nests in populations of seven actual and potential hosts of the common cuckoo Cuculus canorus. Based on these data, we calculate the magnitude of the balancing parasitism rate provided that all eggs lost are a result of recognition errors. Importantly, because eggs are known to disappear from nests for reasons other than erroneous host rejection, our data represent the maximum estimates of such costs. Nonetheless, the disappearance of eggs was a rare event and therefore incurred low costs compared to the high costs of parasitism. Hence, costs as a result of recognition errors are probably of minor importance with respect to opposing selective pressure for the evolution of egg rejection in these hosts. We cannot exclude the possibility that low or intermediate egg rejection rates in some host populations may be caused by spatiotemporal variation in the occurrence of parasitism and gene flow, creating a variable influence of opposing costs as a result of recognition errors and the costs of parasitism.     

Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator

Background  

Novel, uncharacterised proteins represent a challenge in biochemistry and molecular biology. In this report we present an initial functional characterization of human kidney predominant protein, NCU-G1.