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1.  Functional Aspects of the EGF-Induced MAP Kinase Cascade: A Complex Self-Organizing System Approach 
PLoS ONE  2014;9(11):e111612.
The EGF-induced MAP kinase cascade is one of the most important and best characterized networks in intracellular signalling. It has a vital role in the development and maturation of living organisms. However, when deregulated, it is involved in the onset of a number of diseases. Based on a computational model describing a “surface” and an “internalized” parallel route, we use systems biology techniques to characterize aspects of the network’s functional organization. We examine the re-organization of protein groups from low to high external stimulation, define functional groups of proteins within the network, determine the parameter best encoding for input intensity and predict the effect of protein removal to the system’s output response. Extensive functional re-organization of proteins is observed in the lower end of stimulus concentrations. As we move to higher concentrations the variability is less pronounced. 6 functional groups have emerged from a consensus clustering approach, reflecting different dynamical aspects of the network. Mutual information investigation revealed that the maximum activation rate of the two output proteins best encodes for stimulus intensity. Removal of each protein of the network resulted in a range of graded effects, from complete silencing to intense activation. Our results provide a new “vista” of the EGF-induced MAP kinase cascade, from the perspective of complex self-organizing systems. Functional grouping of the proteins reveals an organizational scheme contrasting the current understanding of modular topology. The six identified groups may provide the means to experimentally follow the dynamics of this complex network. Also, the vulnerability analysis approach may be used for the development of novel therapeutic targets in the context of personalized medicine.
PMCID: PMC4221048  PMID: 25372488
2.  Evolutionary conservation of cold-induced antisense RNAs of FLOWERING LOCUS C in Arabidopsis thaliana perennial relatives 
Nature Communications  2014;5:4457.
Antisense RNA (asRNA) COOLAIR is expressed at A. thaliana FLOWERING LOCUS C (FLC) in response to winter temperatures. Its contribution to cold-induced silencing of FLC was proposed but its functional and evolutionary significance remain unclear. Here we identify a highly conserved block containing the COOLAIR first exon and core promoter at the 3′ end of several FLC orthologues. Furthermore, asRNAs related to COOLAIR are expressed at FLC loci in the perennials A. alpina and A. lyrata, although some splicing variants differ from A. thaliana. Study of the A. alpina orthologue, PERPETUAL FLOWERING 1 (PEP1), demonstrates that AaCOOLAIR is induced each winter of the perennial life cycle. Introduction of PEP1 into A. thaliana reveals that AaCOOLAIR cis-elements confer cold-inducibility in this heterologous species while the difference between PEP1 and FLC mRNA patterns depends on both cis-elements and species-specific trans-acting factors. Thus, expression of COOLAIR is highly conserved, supporting its importance in FLC regulation.
FLOWERING LOCUS C (FLC) is thought to control the flowering time of A. thaliana in response to winter temperatures, in a process known as vernalization. Here, the authors suggest that the COOLAIR antisense RNA, which is conserved across plant species, acts to repress the expression of FLC during vernalization.
PMCID: PMC4109010  PMID: 25030056
3.  Analysis of TTG1 function in Arabis alpina 
BMC Plant Biology  2014;14:16.
In Arabidopsis thaliana (A. thaliana) the WD40 protein TRANSPARENT TESTA GLABRA1 (TTG1) controls five traits relevant for the adaptation of plants to environmental changes including the production of proanthocyanidin, anthocyanidin, seed coat mucilage, trichomes and root hairs. The analysis of different Brassicaceae species suggests that the function of TTG1 is conserved within the family.
In this work, we studied the function of TTG1 in Arabis alpina (A. alpina). A comparison of wild type and two Aattg1 alleles revealed that AaTTG1 is involved in the regulation of all five traits. A detailed analysis of the five traits showed striking phenotypic differences between A. alpina and A. thaliana such that trichome formation occurs also at later stages of leaf development and that root hairs form at non-root hair positions.
The evolutionary conservation of the regulation of the five traits by TTG1 on the one hand and the striking phenotypic differences make A. alpina a very interesting genetic model system to study the evolution of TTG1-dependent gene regulatory networks at a functional level.
PMCID: PMC3904473  PMID: 24406039
Arabis alpina; TTG1; Trichomes; Root hairs; Pro-anthocyanidin; Anthocyanidin; Seed coat mucilage
4.  PEP1 of Arabis alpina Is Encoded by Two Overlapping Genes That Contribute to Natural Genetic Variation in Perennial Flowering 
PLoS Genetics  2012;8(12):e1003130.
Higher plants exhibit a variety of different life histories. Annual plants live for less than a year and after flowering produce seeds and senesce. By contrast perennials live for many years, dividing their life cycle into episodes of vegetative growth and flowering. Environmental cues control key check points in both life histories. Genes controlling responses to these cues exhibit natural genetic variation that has been studied most in short-lived annuals. We characterize natural genetic variation conferring differences in the perennial life cycle of Arabis alpina. Previously the accession Pajares was shown to flower after prolonged exposure to cold (vernalization) and only for a limited period before returning to vegetative growth. We describe five accessions of A. alpina that do not require vernalization to flower and flower continuously. Genetic complementation showed that these accessions carry mutant alleles at PERPETUAL FLOWERING 1 (PEP1), which encodes a MADS box transcription factor orthologous to FLOWERING LOCUS C in the annual Arabidopsis thaliana. Each accession carries a different mutation at PEP1, suggesting that such variation has arisen independently many times. Characterization of these alleles demonstrated that in most accessions, including Pajares, the PEP1 locus contains a tandem arrangement of a full length and a partial PEP1 copy, which give rise to two full-length transcripts that are differentially expressed. This complexity contrasts with the single gene present in A. thaliana and might contribute to the more complex expression pattern of PEP1 that is associated with the perennial life-cycle. Our work demonstrates that natural accessions of A. alpina exhibit distinct life histories conferred by differences in PEP1 activity, and that continuous flowering forms have arisen multiple times by inactivation of the floral repressor PEP1. Similar phenotypic variation is found in other herbaceous perennial species, and our results provide a paradigm for how characteristic perennial phenotypes might arise.
Author Summary
Perennial plants live for many years and cycle between flowering and vegetative growth. These stages of the life cycle are often initiated by environmental conditions and occur seasonally. However, many herbaceous perennial species such as strawberry, rose, or Arabis alpina contain varieties that flower continuously irrespective of the seasons. Here we characterize this genetic variation in A. alpina and show that five continuously flowering accessions carry independent mutations in the PERPETUAL FLOWERING 1 (PEP1) gene. These mutations impair the activity of the PEP1 floral repressor causing the plants to flower without requirement for winter cold and to flower continuously. This result has interesting parallels with strawberry and rose, where inactivation of a different floral repressor controlling response to day length gives rise to naturally occurring perpetual flowering forms. We also show that PEP1 in A. alpina has a complex duplicated structure that gives rise to two overlapping transcripts. This arrangement differs from the simple structure of PEP1 orthologues in related annual species, such as FLC of Arabidopsis thaliana, suggesting that duplication of PEP1 might contribute to the complex transcriptional patterns associated with PEP1 function in perennials. Our work provides insight into genetic variation contributing to the perennial life history of plants.
PMCID: PMC3527215  PMID: 23284298
5.  Alcohol Affects the Brain's Resting-State Network in Social Drinkers 
PLoS ONE  2012;7(10):e48641.
Acute alcohol intake is known to enhance inhibition through facilitation of GABAA receptors, which are present in 40% of the synapses all over the brain. Evidence suggests that enhanced GABAergic transmission leads to increased large-scale brain connectivity. Our hypothesis is that acute alcohol intake would increase the functional connectivity of the human brain resting-state network (RSN). To test our hypothesis, electroencephalographic (EEG) measurements were recorded from healthy social drinkers at rest, during eyes-open and eyes-closed sessions, after administering to them an alcoholic beverage or placebo respectively. Salivary alcohol and cortisol served to measure the inebriation and stress levels. By calculating Magnitude Square Coherence (MSC) on standardized Low Resolution Electromagnetic Tomography (sLORETA) solutions, we formed cortical networks over several frequency bands, which were then analyzed in the context of functional connectivity and graph theory. MSC was increased (p<0.05, corrected with False Discovery Rate, FDR corrected) in alpha, beta (eyes-open) and theta bands (eyes-closed) following acute alcohol intake. Graph parameters were accordingly altered in these bands quantifying the effect of alcohol on the structure of brain networks; global efficiency and density were higher and path length was lower during alcohol (vs. placebo, p<0.05). Salivary alcohol concentration was positively correlated with the density of the network in beta band. The degree of specific nodes was elevated following alcohol (vs. placebo). Our findings support the hypothesis that short-term inebriation considerably increases large-scale connectivity in the RSN. The increased baseline functional connectivity can -at least partially- be attributed to the alcohol-induced disruption of the delicate balance between inhibitory and excitatory neurotransmission in favor of inhibitory influences. Thus, it is suggested that short-term inebriation is associated, as expected, to increased GABA transmission and functional connectivity, while long-term alcohol consumption may be linked to exactly the opposite effect.
PMCID: PMC3485329  PMID: 23119078
6.  From muscle research to clinical applications: Do glutamate antagonists aid muscle recovery? 
It has been shown in the rat, that during the first five postnatal days, motoneurons are particularly vulnerable to excitotoxic cell death and glutamate receptors play a significant role in this time-dependent process. Various categories of glutamate blockers (MK-801, Mg, PNQX, DAP-5) have various actions on the respective receptors. Furthermore, the different response between mature and immature motoneurons following injury is attributed to the quantity of glutamate receptors on the cell membrane. The effect of these substances on the recovery of fast and slow muscles after sciatic nerve crush, at critical developmental stages, shows a variable but impressive reversal of the devastating effects on rat muscle properties, which is different between fast and slow muscles. In addition, blocking of NMDA receptors by various substances rescues motoneurons and increases the number of motor units surviving into adulthood.
In this way, glutamate receptor blockers may represent a promising therapeutic approach to retain nerve and muscle function during neurodegenerative events.
PMCID: PMC3666501  PMID: 23738276
excitotoxicity; glutamate antagonists; muscle plasticity; reinnervation; NMDA; AMPA
7.  Contractile properties and movement behaviour in neonatal rats with axotomy, treated with the NMDA antagonist DAP5 
BMC Physiology  2012;12:5.
It is well known that axotomy in the neonatal period causes massive loss of motoneurons, which is reflected in the reduction of the number of motor units and the alteration in muscle properties. This type of neuronal death is attributed to the excessive activation of the ionotropic glutamate receptors (glutamate excitotoxicity). In the present study we investigated the effect of the NMDA antagonist DAP5 [D-2-amino-5-phosphonopentanoic acid] in systemic administration, on muscle properties and on behavioural aspects following peripheral nerve injury.
Wistar rats were subjected to sciatic nerve crush on the second postnatal day. Four experimental groups were included in this study: a) controls (injection of 0.9% NaCl solution) b) crush c) DAP5 treated and d) crush and DAP5 treated. Animals were examined with isometric tension recordings of the fast extensor digitorum longus and the slow soleus muscles, as well as with locomotor tests at four time points, at P14, P21, P28 and adulthood (2 months).
1. Administration of DAP5 alone provoked no apparent adverse effects. 2. In all age groups, animals with crush developed significantly less tension than the controls in both muscles and had a worse performance in locomotor tests (p < 0.01). Crush animals injected with DAP5 were definitely improved as their tension recordings and their locomotor behaviour were significantly improved compared to axotomized ones (p < 0.01). 3. The time course of soleus contraction was not altered by axotomy and the muscle remained slow-contracting in all developmental stages in all experimental groups. EDL, on the other hand, became slower after the crush (p < 0.05). DAP5 administration restored the contraction velocity, even up to the level of control animals 4. Following crush, EDL becomes fatigue resistant after P21 (p < 0.01). Soleus, on the other hand, becomes less fatigue resistant. DAP5 restored the profile in both muscles.
Our results confirm that contractile properties and locomotor behaviour of animals are severely affected by axotomy, with a differential impact on fast contracting muscles. Administration of DAP5 reverses these devastating effects, without any observable side-effects. This agent could possibly show a therapeutic potential in other models of excitotoxic injury as well.
PMCID: PMC3395568  PMID: 22551202
8.  Low-Frequency Fatigue as an Indicator of Eccentric Exercise-Induced Muscle Injury: The Role of Vitamin E 
This study investigates whether vitamin E can attenuate eccentric exercise-induced soleus muscle injury as indicated by the amelioration of in situ isometric force decline following a low-frequency fatigue protocol (stimulation at 4 Hz for 5 min) and the ability of the muscle to recover 3 min after the termination of the fatigue protocol. Adult male Wistar rats were divided into vitamin E-supplemented or placebo-supplemented groups studied at rest, immediately post-exercise or 48 h post-exercise. Daily dl-α-tocopheryl acetate intraperitoneal injections of 100 mg/kg body mass for 5 consecutive days prior to exercise doubled its plasma levels. Fatigue index and recovery index expressed as a percentage of the initial tension. FI at 0 h post- and 48 h post-exercise respectively was 88% ± 4.2% and 89% ± 6.8% in the vitamin E groups versus 76% ± 3% and 80% ± 11% in the placebo groups. RI was 99% ± 3.4% and 100% ± 6% in the vitamin E groups versus 82% ± 3.1% and 84% ± 5.9% in the placebo groups. Complementally to the traditionally recorded maximal force, low-frequency fatigue measures may be beneficial for assessing injury-induced decrease in muscle functionality.
PMCID: PMC3400461  PMID: 22848781
9.  Proteases Inhibition Assessment on PC12 and NGF Treated Cells after Oxygen and Glucose Deprivation Reveals a Distinct Role for Aspartyl Proteases 
PLoS ONE  2011;6(10):e25950.
Hypoxia is a severe stressful condition and induces cell death leading to neuronal loss both to the developing and adult nervous system. Central theme to cellular death is the activation of different classes of proteases such as caspases calpains and cathepsins. In the present study we investigated the involvement of these proteases, in the hypoxia-induced PC12 cell death. Rat PC12 is a model cell line for experimentation relevant to the nervous system and several protocols have been developed for either lethal hypoxia (oxygen and glucose deprivation OGD) or ischemic preconditioning (IPS). Nerve Growth Factor (NGF) treated PC12 differentiate to a sympathetic phenotype, expressing neurites and excitability. Lethal hypoxia was established by exposing undifferentiated and NGF-treated PC12 cells to a mixture of N2/CO2 (93:5%) in DMEM depleted of glucose and sodium pyruvate for 16 h. The involvement of caspases, calpains and lysosomal cathepsins D and E to the cell death induced by lethal OGD was investigated employing protease specific inhibitors such as z-VAD-fmk for the caspases, MDL28170 for the calpains and pepstatin A for the cathepsins D and E. Our findings show that pepstatin A provides statistically significant protection from cell death of both naive and NGF treated PC12 cells exposed to lethal OGD. We propose that apart from the established processes of apoptosis and necrosis that are integral components of lethal OGD, the activation of cathepsins D and E launches additional cell death pathways in which these proteases are key partners.
PMCID: PMC3196512  PMID: 22028798
10.  Morphological, histochemical, and interstitial pressure changes in the tibialis anterior muscle before and after aortofemoral bypass in patients with peripheral arterial occlusive disease 
Morphological and electrophysiological studies of ischemic muscles in peripheral arterial disease disclosed evidence of denervation and fibre atrophy. The purpose of the present study is to describe morphological changes in ischemic muscles before and after reperfusion surgery in patients with peripheral occlusive arterial disease, and to provide an insight into the effect of reperfusion on the histochemistry of the reperfused muscle.
Muscle biopsies were obtained from the tibialis anterior of 9 patients with chronic peripheral arterial occlusive disease of the lower extremities, before and after aortofemoral bypass, in order to evaluate the extent and type of muscle fibre changes during ischemia and after revascularization. Fibre type content and muscle fibre areas were quantified using standard histological and histochemical methods and morphometric analysis. Each patient underwent concentric needle electromyography, nerve conduction velocity studies, and interstitial pressure measurements.
Preoperatively all patients showed muscle fibre atrophy of both types, type II fibre area being more affected. The mean fibre cross sectional area of type I was 3,745 μm2 and of type II 4,654 μm2 . Fibre-type grouping, great variation in fibre size and angular fibres were indicative of chronic dennervation-reinnervation, in the absence of any clinical evidence of a neuropathic process. Seven days after the reperfusion the areas of both fibre types were even more reduced, being 3,086 μm2 for type I and 4,009 μm2 for type II, the proportion of type I fibres, and the interstitial pressure of tibialis anterior were increased.
The findings suggest that chronic ischemia of the leg muscles causes compensatory histochemical changes in muscle fibres resulting from muscle hypoxia, and chronic dennervation-reinnervation changes, resulting possibly from ischemic neuropathy. Reperfusion seems to bring the oxidative capacity of the previously ischemic muscle closer to normal.
PMCID: PMC89010  PMID: 11895571

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