Cellular Mechanisms of Myocardial Depression in Porcine Septic Shock.

The complex pathogenesis of sepsis and septic shock involves myocardial depression, the pathophysiology of which, however, remains unclear. In this study, cellular mechanisms of myocardial depression were addressed in a clinically relevant, large animal (porcine) model of sepsis and septic shock. Sepsis was induced by fecal peritonitis in eight anesthetized, mechanically ventilated, and instrumented pigs of both sexes and continued for 24 h. In eight control pigs, an identical experiment but without sepsis induction was performed. In vitro analysis of cardiac function included measurements of action potentials and contractions in the right ventricle trabeculae, measurements of sarcomeric contractions, calcium transients and calcium current in isolated cardiac myocytes, and analysis of mitochondrial respiration by ultrasensitive oxygraphy. Increased values of modified sequential organ failure assessment score and serum lactate levels documented the development of sepsis/septic shock, accompanied by hyperdynamic circulation with high heart rate, increased cardiac output, peripheral vasodilation, and decreased stroke volume. In septic trabeculae, action potential duration was shortened and contraction force reduced. In septic cardiac myocytes, sarcomeric contractions, calcium transients, and L-type calcium current were all suppressed. Similar relaxation trajectory of the intracellular calcium-cell length phase-plane diagram indicated unchanged calcium responsiveness of myofilaments. Mitochondrial respiration was diminished through inhibition of Complex II and Complex IV. Defective calcium handling with reduced calcium current and transients, together with inhibition of mitochondrial respiration, appears to represent the dominant cellular mechanisms of myocardial depression in porcine septic shock.

Stabilizing selection on genome size in a population of Festuca pallens under conditions of intensive intraspecific competition.

*Stabilizing selection is a key evolutionary mechanism for which there is relatively little experimental evidence. To date, stabilizing selection has never been observed at the whole-genome level. *We tested the effect of selection on genome size in a field experiment using seeds collected in a population of Festuca pallens with a highly variable genome size. Using flow cytometry, we measured the genome size in germinating seedlings and juvenile plants grown with or without high intraspecific competition (908 individuals). Above-ground biomass and leaf number were used as measurements of individual vegetative performance. The possible confounding effect of seed weight was controlled for in a separate experiment. *Growth under high competition had a significant stabilizing effect on genome size. Because no relationship was observed between genome size and vegetative performance, we assume that the elimination of plants with extreme genome sizes was the result of decreased survival as a consequence of some unrecognized stress. *Our results indicate that genome size may be under direct selection. The equal disadvantaging of either large or small genomes indicates that the selection for optimum genome size in species may be fully context dependent. This study demonstrates the power of competition experiments for the detection of weak selection processes.

An interspecific hybrid as a tool to study phylogenetic relationships in plants using the GISH technique.

We established a new auxiliary phylogenetic approach based on genomic in situ hybridization technique (GISH). We used an interspecific hybrid Silene latifolia x Silene viscosa to compare two different genomes simultaneously on one slide. By using GISH with genomic DNA from another closely related species as a probe, we directly compared the level of relatedness between the genomes of the studied species and parental species. This experimental design enabled us to approximately estimate evolutionary relationships between the genome of tested plant species and genomes of both parental species of the hybrid by using the ratio of intensities of fluorescence signals. We tested this technique in various Silene species and the results were in accordance with the topology of the phylogenetic tree we constructed based on rDNA sequences. The results were also well correlated with phylogenetic distances between species that we estimated from an rDNA-based phylogenetic tree. Our experimental approach could help to improve tree topology and serve as a useful complementary tool in molecular phylogenetic studies in related species.

Karyological analysis of an interspecific hybrid between the dioecious Silene latifolia and the hermaphroditic Silene viscosa.

The genus Silene is a good model for studying evolution of the sex chromosomes, since it includes species that are hermaphroditic and dioecious, while maintain a basic chromosome number of 2n = 24. For some combinations of Silene species it is possible to construct interspecific hybrids. Here, we present a detailed karyological analysis of a hybrid between the dioecious Silene latifolia as the maternal plant and a related species, hermaphroditic Silene viscosa, used as a pollen partner. Using genomic probes (the genomic in situ hybridization (GISH) technique), we were able to clearly discriminate parental genomes and to show that they are largely separated in distinct nuclear domains. Molecular GISH and fluorescence in situ hybridization (FISH) markers document that the hybrid genome of somatic cells was strictly additive and stable, and that it had 12 chromosomes originating from each parent, including the only X chromosome of S. latifolia. Meiotic analysis revealed that, although related, respective parental chromosomes did not pair or paired only partially, which resulted in frequent chromosome abnormalities such as bridges and irregular non-disjunctions. GISH and FISH markers clearly document that the larger genome of S. latifolia and its largest chromosome component, the X chromosome, were mostly employed in chromosome lagging and misdivision.

Determinants of substrate specificity in omega-aminotransferases.

Ornithine aminotransferase and 4-aminobutyrate aminotransferase are related pyridoxal phosphate-dependent enzymes having different substrate specificities. The atomic structures of these enzymes have shown (i) that active site differences are limited to the steric positions occupied by two tyrosine residues in ornithine aminotransferase and (ii) that, uniquely among related, structurally characterized aminotransferases, the conserved arginine that binds the alpha-carboxylate of alpha-amino acids interacts tightly with a glutamate residue. To determine the contribution of these residues to the specificities of the enzymes, we analyzed site-directed mutants of ornithine aminotransferase by rapid reaction kinetics, x-ray crystallography, and 13C NMR spectroscopy. Mutation of one tyrosine (Tyr-85) to isoleucine, as found in aminobutyrate aminotransferase, decreased the rate of the reaction of the enzyme with ornithine 1000-fold and increased that with 4-aminobutyrate 16-fold, indicating that Tyr-85 is a major determinant of specificity toward ornithine. Unexpectedly, the limiting rate of the second half of the reaction, conversion of ketoglutarate to glutamate, was greatly increased, although the kinetics of the reverse reaction were unaffected. A mutant in which the glutamate (Glu-235) that interacts with the conserved arginine was replaced by alanine retained its regiospecificity for the delta-amino group of ornithine, but the glutamate reaction was enhanced 650-fold, whereas only a 5-fold enhancement of the ketoglutarate reaction rate resulted. A model is proposed in which conversion of the enzyme to its pyridoxamine phosphate form disrupts the internal glutamate-arginine interaction, thus enabling ketoglutarate but not glutamate to be a good substrate.

The inter-specific hybrid Silene latifolia x S. viscosa reveals early events of sex chromosome evolution.

The dioecious plant species Silene latifolia has a sex determination mechanism based on an active Y chromosome. Here, we used inter-specific hybrids in the genus Silene to study the effects of gene complexes on the Y chromosome. If the function of Y-linked genes has been maintained in the same state as in the hermaphrodite progenitor species, it should be possible to substitute such genes by genes coming from a related hermaphrodite species. In the inter-specific hybrid, S. latifolia x S. viscosa, anthers indeed develop far beyond the early bilobal stage characteristic of XX S. latifolia female plants. The S. viscosa genome can thus replace the key sex determination gene whose absence abolishes early stamen development in females (loss of the stamen-promoting function, SPF), so that hybrid plants are morphologically hermaphrodite. However, the hybrids have two anther development defects, loss of adhesion of the tapetum to the endothecium, and precocious endothecium maturation. Both these defects were also found in independent Y-chromosome deletion mutants of S. latifolia. The data support the hypothesis that the evolution of complete gender dimorphism from hermaphroditism involved a major largely recessive male-sterility factor that created females, and the appearance of new, dominant genes on the Y chromosome, including both the well-documented gynoecium-suppressing factor, and two other Y specific genes promoting anther development.