Visible and "cryptic" segregation of parental chromosomes in embryonic stem hybrid cells

Chromosome segregation of the parental chromosomes was studied in 20 interspecific hybrid clones obtained by fusion of Mus musculus embryonic stem cells with Mus caroli splenocytes. FISH analysis with labeled species specific probes and microsatellite markers was used for identification of the parental chromosomes. Cytogenetic analysis has shown significant intra- and interclonal variability in chromosome numbers and ratios of the parental chromosomes in the hybrid cells: six clones contained all M. caroli chromosomes, nine clones showed moderate segregation of M. caroli chromosomes (from 1 to 7), and five clones showed extensive loss of M. caroli chromosomes (from 12 to complete loss of all M. caroli autosomes). Both methods demonstrated "cryptic" segregation of the somatic partner chromosomes. For instance, five clones with near-tetraploid chromosome sets contained only few M. caroli chromosomes (from 1 to 8). The data obtained suggest that the tetraploid chromosome set per se is not a sufficient criterion for conclusion on the absence of chromosome loss in the hybrid cells. Note that "cryptic" chromosome segregation occurred at a high frequency in the examined hybrid clones. Thus, "cryptic" segregation should be borne in mind for assessing pluripotency and genome reprogramming of embryonic stem hybrid cells.     

Structural basis for autoinhibition and mutational activation of eukaryotic initiation factor 2alpha protein kinase GCN2

The GCN2 protein kinase coordinates protein synthesis with levels of amino acid stores by phosphorylating eukaryotic translation initiation factor 2. The autoinhibited form of GCN2 is activated in cells starved of amino acids by binding of uncharged tRNA to a histidyl-tRNA synthetase-like domain. Replacement of Arg-794 with Gly in the PK domain (R794G) activates GCN2 independently of tRNA binding. Crystal structures of the GCN2 protein kinase domain have been determined for wild-type and R794G mutant forms in the apo state and bound to ATP/AMPPNP. These structures reveal that GCN2 autoinhibition results from stabilization of a closed conformation that restricts ATP binding. The R794G mutant shows increased flexibility in the hinge region connecting the N- and C-lobes, resulting from loss of multiple interactions involving Arg794. This conformational change is associated with intradomain movement that enhances ATP binding and hydrolysis. We propose that intramolecular interactions following tRNA binding remodel the hinge region in a manner similar to the mechanism of enzyme activation elicited by the R794G mutation.     

Fluorescent analysis of irradiation-induced changes in chromatin state

A fluorescent technique with Hoechst-33258 and acridine orange staining was used to assess changes in chromatin state induced by radiation. Fluorochromes with different modes of binding to DNA were chosen. In T lymphocytes chronic irradiation caused a rearrangement of binding between nonhiston proteins and lipids accompanied by conformational changes in DNA, resulting in chromatin condensation. The decrease in fluorescence probably resulted from a reduction in the number of sites accessible for dye binding. After acute irradiation, the fluorescence intensity decreases predominantly due to double-strand breaks.     

Effects of Tail Clipping on Larval Performance and Tail Regeneration Rates in the Near Eastern Fire Salamander,Salamandra infraimmaculata

Tail-tip clipping is a common technique for collecting tissue samples from amphibian larvae and adults. Surprisingly, studies of this invasive sampling procedure or of natural tail clipping – i.e., bites inflicted by predators including conspecifics - on the performance and fitness of aquatic larval stages of urodeles are scarce. We conducted two studies in which we assessed the effects of posterior tail clipping (~30 percent of tail) on Near Eastern fire salamander (Salamandra infraimmaculata) larvae. In a laboratory study, we checked regeneration rates of posterior tail-tip clipping at different ages. Regeneration rates were hump-shaped, peaking at the age of ~30 days and then decreasing. This variation in tail regeneration rates suggests tradeoffs in resource allocation between regeneration and somatic growth during early and advanced development. In an outdoor artificial pond experiment, under constant larval densities, we assessed how tail clipping of newborn larvae affects survival to, time to, and size at metamorphosis. Repeated measures ANOVA on mean larval survival per pond revealed no effect of tail clipping. Tail clipping had correspondingly no effect on larval growth and development expressed in size (mass and snout-vent length) at, and time to, metamorphosis. We conclude that despite the given variation in tail regeneration rates throughout larval ontogeny, clipping of 30% percent of the posterior tail area seems to have no adverse effects on larval fitness and survival. We suggest that future use of this imperative tool for the study of amphibian should take into account larval developmental stage during the time of application and not just the relative size of the clipped tail sample.     

Derivatives of the cationic plant alkaloids berberine and palmatine amplify protonophorous activity of fatty acids in model membranes and mitochondria

Previously it has been shown by our group that berberine and palmatine, penetrating cations of plant origin, when conjugated with plastoquinone (SkQBerb and SkQPalm), can accumulate in isolated mitochondria or in mitochondria of living cells and effectively protect them from oxidative damage. In the present work, we demonstrate that SkQBerb, SkQPalm, and their analogs lacking the plastoquinone moiety (C₁₀Berb and C₁₀Palm) operate as mitochondria-targeted compounds facilitating protonophorous effect of free fatty acids. These compounds induce proton transport mediated by small concentrations of added fatty acids both in planar and liposomal model lipid membranes. In mitochondria, such an effect can be carried out by endogenous fatty acids and the adenine nucleotide translocase.     

Determination of reactive oxygen and nitrogen species in rat aorta using the dichlorofluorescein assay

A method for the determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in macroscopic sections of vessels has been developed on the basis of the dichlorofluorescein (DCF) assay. DCF was measured by fluorescence in extracts of vessels. The main artifact of the method is the oxidation of dichlorodihydrofluorescein (DCFH₂) which is released from vessels together with DCF during the extraction procedure. This problem was resolved by decreasing pH during the extraction. The optimal conditions and the time for aorta incubation with DCFH₂-DA and for the extraction of DCF from aorta have been determined. The ROS/RNS production in different aorta segments and the dependence of ROS/RNS production on rat age have been studied. It was shown that thoracic aorta sections produced the same amounts of ROS/RNS and the intermediate between the thoracic and the abdominal aorta part produced ROS and RNS by 14% more than the thoracic aorta. It was found that ROS/RNS production in aorta increases with rat age: the doubling time of ROS/RNS production rate is 113 days from birth.     

Systematic and evolutionary insights derived from mtDNA COI barcode diversity in the Decapoda (Crustacea: Malacostraca)

Background

Decapods are the most recognizable of all crustaceans and comprise a dominant group of benthic invertebrates of the continental shelf and slope, including many species of economic importance. Of the 17635 morphologically described Decapoda species, only 5.4% are represented by COI barcode region sequences. It therefore remains a challenge to compile regional databases that identify and analyse the extent and patterns of decapod diversity throughout the world.

Methodology/Principal Findings

We contributed 101 decapod species from the North East Atlantic, the Gulf of Cadiz and the Mediterranean Sea, of which 81 species represent novel COI records. Within the newly-generated dataset, 3.6% of the species barcodes conflicted with the assigned morphological taxonomic identification, highlighting both the apparent taxonomic ambiguity among certain groups, and the need for an accelerated and independent taxonomic approach. Using the combined COI barcode projects from the Barcode of Life Database, we provide the most comprehensive COI data set so far examined for the Order (1572 sequences of 528 species, 213 genera, and 67 families). Patterns within families show a general predicted molecular hierarchy, but the scale of divergence at each taxonomic level appears to vary extensively between families. The range values of mean K2P distance observed were: within species 0.285% to 1.375%, within genus 6.376% to 20.924% and within family 11.392% to 25.617%. Nucleotide composition varied greatly across decapods, ranging from 30.8 % to 49.4 % GC content.

Conclusions/Significance

Decapod biological diversity was quantified by identifying putative cryptic species allowing a rapid assessment of taxon diversity in groups that have until now received limited morphological and systematic examination. We highlight taxonomic groups or species with unusual nucleotide composition or evolutionary rates. Such data are relevant to strategies for conservation of existing decapod biodiversity, as well as elucidating the mechanisms and constraints shaping the patterns observed.     

Preimplantation exposure of mouse embryos to palmitic acid results in fetal growth restriction followed by catch-up growth in the offspring

Free fatty acids (FFAs) are energy substrates for many cell types, but in excess, some FFAs can accumulate in nonadipose cells, inducing apoptosis. Also known as lipotoxicity, this phenomenon may play a role in the development of obesity-related disease. Obesity is common among reproductive age women and is associated with adverse pregnancy and fetal outcomes; however, little is known about the effects of excess FFAs on embryos and subsequent fetal development. To address this knowledge gap, murine blastocysts were cultured in excess palmitic acid (PA), the most abundant saturated FFA in human serum, and ovarian follicular fluid. Targets susceptible to aberrations in maternal physiology, including embryonic IGF1 receptor (IGF1R) expression, glutamic pyruvate transaminase (GPT2) activity, and nuclei count, were measured. PA-exposed blastocysts demonstrated altered IGF1R expression, increased GPT2 activity, and decreased nuclei count. Trophoblast stem cells derived from preimplantation embryos were also cultured in PA. Cells exposed to increasing doses of PA demonstrated increased apoptosis and decreased proliferation. To demonstrate long-term effects of brief PA exposure, blastocysts cultured for 30 h in PA were transferred into foster mice, and pregnancies followed through Embryonic Day (ED)14.5 or delivery. Fetuses resulting from PA-exposed blastocysts were smaller than controls at ED14.5. Delivered pups were also smaller but demonstrated catch-up growth and ultimately surpassed control pups in weight. Altogether, our data suggest brief PA exposure results in altered embryonic metabolism and growth, with lasting adverse effects on offspring, providing further insight into the pathophysiology of maternal obesity.     

Abnormal Expression of Cerebrospinal Fluid Cation Chloride Cotransporters in Patients with Rett Syndrome

Objective

Rett Syndrome is a progressive neurodevelopmental disorder caused mainly by mutations in the gene encoding methyl-CpG-binding protein 2. The relevance of MeCP2 for GABAergic function was previously documented in animal models. In these models, animals show deficits in brain-derived neurotrophic factor, which is thought to contribute to the pathogenesis of this disease. Neuronal Cation Chloride Cotransporters (CCCs) play a key role in GABAergic neuronal maturation, and brain-derived neurotrophic factor is implicated in the regulation of CCCs expression during development. Our aim was to analyse the expression of two relevant CCCs, NKCC1 and KCC2, in the cerebrospinal fluid of Rett syndrome patients and compare it with a normal control group.

Methods

The presence of bumetanide sensitive NKCC1 and KCC2 was analysed in cerebrospinal fluid samples from a control pediatric population (1 day to 14 years of life) and from Rett syndrome patients (2 to 19 years of life), by immunoblot analysis.

Results

Both proteins were detected in the cerebrospinal fluid and their levels are higher in the early postnatal period. However, Rett syndrome patients showed significantly reduced levels of KCC2 and KCC2/NKCC1 ratio when compared to the control group.

Conclusions

Reduced KCC2/NKCC1 ratio in the cerebrospinal fluid of Rett Syndrome patients suggests a disturbed process of GABAergic neuronal maturation and open up a new therapeutic perspective.