The effect of valinomycin in fibroblasts from patients with fatty acid oxidation disorders

Disorders of the carnitine cycle and of the beta oxidation spiral impair the ability to obtain energy from fats at time of fasting and stress. This can result in hypoketotic hypoglycemia, cardiomyopathy, cardiac arrhythmia and other chronic medical problems. The in vitro study of fibroblasts from patients with these conditions is impaired by their limited oxidative capacity. Here we evaluate the capacity of valinomycin, a potassium ionophore that increases mitochondrial respiration, to increase the oxidation of fatty acids in cells from patients with inherited fatty acid oxidation defects. The addition of valinomycin to fibroblasts decreased the accumulation of the lipophilic cation tetraphenylphosphonium (TPP⁺) at low concentrations due to the dissipation of the mitochondrial membrane potential. At higher doses, valinomycin increased TPP⁺ accumulation due to the increased potassium permeability of the plasma membrane and subsequent cellular hyperpolarization. The incubation of normal fibroblasts with valinomycin increased [¹⁴C]-palmitate oxidation (measured as [¹⁴C]O₂ release) in a dose-dependent manner. By contrast, valinomycin failed to increase palmitate oxidation in fibroblasts from patients with very long chain acyl CoA dehydrogenase (VLCAD) deficiency. This was not observed in fibroblasts from patients heterozygous for this condition. These results indicate that valinomycin can increase fatty acid oxidation in normal fibroblasts and could be useful to differentiate heterozygotes from patients affected with VLCAD deficiency.     

The role of climate,water and biotic interactions in shaping biodiversity patterns in arid environments across spatial scales

Aim

Desert ecosystems, with their harsh environmental conditions, hold the key to understanding the responses of biodiversity to climate change. As desert community structure is influenced by processes acting at different spatial scales, studies combining multiple scales are essential for understanding the conservation requirements of desert biota. We investigated the role of environmental variables and biotic interactions in shaping broad and fine‐scale patterns of diversity and distribution of bats in arid environments to understand how the expansion of nondesert species can affect the long‐term conservation of desert biodiversity.

Location

Levant, Eastern Mediterranean.

Methods

We combine species distribution modelling and niche overlap statistics with a statistical model selection approach to integrate interspecific interactions into broadscale distribution models and fine‐scale analysis of ecological requirements. We focus on competition between desert bats and mesic species that recently expanded their distribution into arid environment following anthropogenic land‐use changes.

Results

We show that both climate and water availability limit bat distributions and diversity across spatial scales. The broadscale distribution of bats was determined by proximity to water and high temperatures, although the latter did not affect the distribution of mesic species. At the fine‐scale, high levels of bat activity and diversity were associated with increased water availability and warmer periods. Desert species were strongly associated with warmer and drier desert types. Range and niche overlap were high among potential competitors, but coexistence was facilitated through fine‐scale spatial partitioning of water resources.

Main conclusions

Adaptations to drier and warmer conditions allow desert‐obligate species to prevail in more arid environments. However, this competitive advantage may disappear as anthropogenic activities encroach further into desert habitats. We conclude that reduced water availability in arid environments under future climate change projections pose a major threat to desert wildlife because it can affect survival and reproductive success and may increase competition over remaining water resources.     

Identification of male-specific amh duplication,sexually differentially expressed genes and microRNAs at early embryonic development of Nile tilapia (Oreochromis niloticus)

Background

The probable influence of genes and the environment on sex determination in Nile tilapia suggests that it should be regarded as a complex trait. Detection of sex determination genes in tilapia has both scientific and commercial importance. The main objective was to detect genes and microRNAs that were differentially expressed by gender in early embryonic development.

Results

Artificial fertilization of Oreochromis niloticus XX females with either sex-reversed ΔXX males or genetically-modified YY ‘supermales’ resulted in all-female and all-male embryos, respectively. RNA of pools of all-female and all-male embryos at 2, 5 and 9 dpf were used as template for a custom Agilent eArray hybridization and next generation sequencing. Fifty-nine genes differentially expressed between genders were identified by a false discovery rate of p < 0.05. The most overexpressed genes were amh and tspan8 in males, and cr/20β-hsd, gpa33, rtn4ipl and zp3 in females (p < 1 × 10⁻⁹). Validation of gene expression using qPCR in embryos and gonads indicated copy number variation in tspan8, gpa33, cr/20β-hsd and amh. Sequencing of amh identified a male-specific duplication of this gene, denoted amhy, differing from the sequence of amh by a 233 bp deletion on exonVII, hence lacking the capability to encode the protein motif that binds to the transforming growth factor beta receptor (TGF-β domain). amh and amhy segregated in the mapping family in full concordance with SD-linked marker on LG23 signifying the QTL for SD. We discovered 831 microRNAs in tilapia embryos of which nine had sexually dimorphic expression patterns by a false discovery rate of p < 0.05. An up-regulated microRNA in males, pma-mir-4585, was characterized with all six predicted target genes including cr/20β-hsd, down-regulated in males.

Conclusions

This study reports the first discovery of sexually differentially expressed genes and microRNAs at a very early stage of tilapia embryonic development, i.e. from 2 dpf. Genes with sexually differential expression patterns are enriched for copy number variation. A novel male-specific duplication of amh, denoted amhy, lacking the TGF-β domain was identified and mapped to the QTL region on LG23 for SD, thus indicating its potential role in SD.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-774) contains supplementary material, which is available to authorized users.     

WASp Family Verprolin-homologous Protein-2 (WAVE2) and Wiskott-Aldrich Syndrome Protein (WASp) Engage in Distinct Downstream Signaling Interactions at the T Cell Antigen Receptor Site

T cell antigen receptor (TCR) engagement has been shown to activate pathways leading to actin cytoskeletal polymerization and reorganization, which are essential for lymphocyte activation and function. Several actin regulatory proteins were implicated in regulating the actin machinery, such as members of the Wiskott-Aldrich syndrome protein (WASp) family. These include WASp and the WASp family verprolin-homologous protein-2 (WAVE2). Although WASp and WAVE2 share several structural features, the precise regulatory mechanisms and potential redundancy between them have not been fully characterized. Specifically, unlike WASp, the dynamic molecular interactions that regulate WAVE2 recruitment to the cell membrane and specifically to the TCR signaling complex are largely unknown. Here, we identify the molecular mechanism that controls the recruitment of WAVE2 in comparison with WASp. Using fluorescence resonance energy transfer (FRET) and novel triple-color FRET (3FRET) technology, we demonstrate how WAVE2 signaling complexes are dynamically regulated during lymphocyte activation in vivo. We show that, similar to WASp, WAVE2 recruitment to the TCR site depends on protein-tyrosine kinase, ZAP-70, and the adaptors LAT, SLP-76, and Nck. However, in contrast to WASp, WAVE2 leaves this signaling complex and migrates peripherally together with vinculin to the membrane leading edge. Our experiments demonstrate that WASp and WAVE2 differ in their dynamics and their associated proteins. Thus, this study reveals the differential mechanisms regulating the function of these cytoskeletal proteins.     

Combined Effects of CO2 and Light on the N2-Fixing Cyanobacterium Trichodesmium IMS101: A Mechanistic View

The marine diazotrophic cyanobacterium Trichodesmium responds to elevated atmospheric CO₂ partial pressure (pCO₂) with higher N₂ fixation and growth rates. To unveil the underlying mechanisms, we examined the combined influence of pCO₂ (150 and 900 μatm) and light (50 and 200 μmol photons m⁻² s⁻¹) on Trichodesmium IMS101. We expand on a complementary study that demonstrated that while elevated pCO₂ enhanced N₂ fixation and growth, oxygen evolution and carbon fixation increased mainly as a response to high light. Here, we investigated changes in the photosynthetic fluorescence parameters of photosystem II, in ratios of the photosynthetic units (photosystem I:photosystem II), and in the pool sizes of key proteins involved in the fixation of carbon and nitrogen as well as their subsequent assimilation. We show that the combined elevation in pCO₂ and light controlled the operation of the CO₂-concentrating mechanism and enhanced protein activity without increasing their pool size. Moreover, elevated pCO₂ and high light decreased the amounts of several key proteins (NifH, PsbA, and PsaC), while amounts of AtpB and RbcL did not significantly change. Reduced investment in protein biosynthesis, without notably changing photosynthetic fluxes, could free up energy that can be reallocated to increase N₂ fixation and growth at elevated pCO₂ and light. We suggest that changes in the redox state of the photosynthetic electron transport chain and posttranslational regulation of key proteins mediate the high flexibility in resources and energy allocation in Trichodesmium. This strategy should enable Trichodesmium to flourish in future surface oceans characterized by elevated pCO₂, higher temperatures, and high light.The marine filamentous N₂-fixing (diazotrophic) cyanobacteria Trichodesmium spp. bloom extensively in the oligotrophic subtropical and tropical oceans (Carpenter and Capone, 2008). Trichodesmium contributes 25% to 50% of the estimated rates of N₂ fixation in these areas, where the new nitrogen inputs stimulate carbon and nitrogen cycling (Capone and Subramaniam, 2005; Mahaffey et al., 2005). The increases in atmospheric CO₂ partial pressure (pCO₂) and the subsequent impacts on ocean acidification are predicted to influence diazotrophs and specifically Trichodesmium.The reported sensitivity of Trichodesmium to changes in pCO₂ prompted further investigation into the cellular responses and underlying mechanisms, specifically when combined with other environmental parameters such as temperature, nutrient availability, and light. Elevated pCO₂ significantly increased growth and N₂ fixation rates of Trichodesmium cultures (Barcelos é Ramos et al., 2007; Hutchins et al., 2007; Levitan et al., 2007, 2010). The physiological response was also characterized by changes in inorganic carbon acquisition, limited flexibility of carbon-nitrogen ratios, and conservation of photosynthetic activities with increased pCO₂. These manifestations suggested that ATP and reductants [ferredoxin, NAD(P)H] are reallocated in the cells (Levitan et al., 2007, 2010; Kranz et al., 2009, 2010).In Trichodesmium, as in all cyanobacteria, the metabolic pathways of respiration and photosynthesis share several cellular complexes/proteins such as the plastoquinone (PQ) pool, succinate dehydrogenase, and ferredoxin (Fig. 1; Kana, 1993; Bergman et al., 1997; Lin et al., 1998). Energetic currencies [reduced ferredoxin, ATP, NAD(P)H] are also shared and can be allocated and utilized according to cellular requirements. N₂ fixation by nitrogenase and the subsequent assimilation of NH₄⁺ by Gln synthetase requires carbon skeletons from the tricarboxylic acid reactions. Moreover, linear and pseudocyclic photosynthesis can also generate additional ATP and reductants essential for N₂ fixation (Fig. 1; Berman-Frank et al., 2001).Open in a separate windowFigure 1.Schematic representation of major cellular complexes involved in energy flow [electron, ATP, NAD(P)H, carbon skeletons] in Trichodesmium IMS101. Dashed arrows represent movement direction of electrons, and solid arrows represent directions of protons, ATP, and NAD(P)H. Measured protein subunits are represented by gray diamonds. See Kranz et al. (2010) for measurements of O₂ evolution, inorganic carbon fixation, and fluxes of N₂ fixation.To understand the regulation of these metabolic pathways in Trichodesmium under varying pCO₂ levels and light intensities, we designed an experiment to characterize changes in the fluxes of carbon, nitrogen, and oxygen (O₂), related protein pool sizes, and variable fluorescence parameters of PSII. Elevated atmospheric pCO₂ combined with enhanced sea surface temperatures are forecast to stabilize thermal stratification, resulting in a shallower, more acidified, upper mixed layer characterized by higher mean light intensities (Doney, 2006). Thus, Trichodesmium IMS101 cultures were acclimated to past and future pCO₂ levels (150 and 900 μatm) at low and high light (50 and 200 μmol photons m⁻² s⁻¹).In the first part of this combined report (Kranz et al., 2010), we examined the physiological responses to the different acclimation conditions. The combination of elevated pCO₂ and light enhanced the production of particulate organic carbon and nitrogen (270% and 390% increase, respectively) as well as growth rates (180% increase; percentages are calculated from Kranz et al., 2010). Generally, the pCO₂-dependent stimulation was higher in cultures acclimated to low light. The pCO₂ effect was also reflected in other measured physiological parameters, particularly the diel patterns of N₂ fixation and the integrated N₂ fixation rates during the day, which increased approximately 30-fold between the low-pCO₂/low-light and the high-pCO₂/high-light acclimations (Kranz et al., 2010). While at high light, elevated pCO₂ extended the period of high N₂ fixation, which lasted from 5 h after the onset of light throughout the end of the photoperiod, the high-pCO₂ contribution to the integrated N₂ fixation was more significant at low light (Kranz et al., 2010). Light, but not pCO₂, influenced gross photosynthesis as measured by PSII O₂ evolution, which increased by approximately 250% in high-light-acclimated cultures. To supply the Calvin cycle with sufficient CO₂, Trichodesmium possesses a CO₂-concentrating mechanism mainly based on HCO₃⁻ uptake (Kranz et al., 2009, 2010). When Trichodesmium was acclimated to elevated pCO₂ (900 μatm), a decline in the cellular affinity to dissolved inorganic carbon was observed (Kranz et al., 2009), while the specific uptake of CO₂ showed a 9-fold increase between the low-pCO₂/low-light and the high-pCO₂/high-light acclimations (Kranz et al., 2010).Proteins are fundamental cellular components that influence the underlying mechanisms subsequently reflected in the cells’ physiology. In this study, we extend the experimental results presented by Kranz et al. (2010) by examining the influence of pCO₂ at different light regimes on the photosynthetic fluorescence parameters of PSII and on the pool sizes of key proteins involved in carbon and nitrogen fixation and their subsequent assimilation processes.     

Reversible Cysteine Acylation Regulates the Activity of Human Palmitoyl-Protein Thioesterase 1 (PPT1)

Mutations in the depalmitoylating enzyme gene, PPT1, cause the infantile form of Neuronal Ceroid Lipofuscinosis (NCL), an early onset neurodegenerative disease. During recent years there have been different therapeutic attempts including enzyme replacement. Here we show that PPT1 is palmitoylated in vivo and is a substrate for two palmitoylating enzymes, DHHC3 and DHHC7. The palmitoylated protein is detected in both cell lysates and medium. The presence of PPT1 with palmitoylated signal peptide in the cell medium suggests that a subset of the protein is secreted by a nonconventional mechanism. Using a mutant form of PPT1, C6S, which was not palmitoylated, we further demonstrate that palmitoylation does not affect intracellular localization but rather that the unpalmitoylated form enhanced the depalmitoylation activity of the protein. The calculated Vmax of the enzyme was significantly affected by the palmitoylation, suggesting that the addition of a palmitate group is reminiscent of adding a noncompetitive inhibitor. Thus, we reveal the existence of a positive feedback loop, where palmitoylation of PPT1 results in decreased activity and subsequent elevation in the amount of palmitoylated proteins. This positive feedback loop is likely to initiate a vicious cycle, which will enhance disease progression. The understanding of this process may facilitate enzyme replacement strategies.     

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at ≥6 months after birth. We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.     

Analysing the origin of long-range interactions in proteins using lattice models

Background  

Long-range communication is very common in proteins but the physical basis of this phenomenon remains unclear. In order to gain insight into this problem, we decided to explore whether long-range interactions exist in lattice models of proteins. Lattice models of proteins have proven to capture some of the basic properties of real proteins and, thus, can be used for elucidating general principles of protein stability and folding.     

DNase I sensitivity of Microtus agrestis active,inactive and reactivated X chromosomes in mouse-Microtus cell hybrids

We isolated Microtus agrestis-mouse somatic cell hybrid clones which had retained either the active or the inactive M. agrestis X chromosome. In both hybrid clones the X chromosomes retained their original chromatin conformation as studied by the in situ nick translation technique — the active X chromosome retained its high sensitivity to DNase I while the inactive one remained insensitive. A clone in which the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene had been spontaneously reactivated was isolated from the hybrid containing the inactive X chromosome. The in situ nick translation technique was used to study possible DNA conformation changes in the euchromatin of the inactive X chromosome with special reference to the reactivated HPRT locus. We found that the euchromatin in this X chromosome exhibited the same low sensitivity to DNase I as is characteristic of the inactive X chromosome.Professor Marcus passed away on 2 January 1987