EVOLUTION OF SEX DETERMINATION SYSTEMS WITH HETEROGAMETIC MALES AND FEMALES IN SILENE

The plant genus Silene has become a model for evolutionary studies of sex chromosomes and sex‐determining mechanisms. A recent study performed in Silene colpophylla showed that dioecy and the sex chromosomes in this species evolved independently from those in Silene latifolia, the most widely studied dioecious Silene species. The results of this study show that the sex‐determining system in Silene otites, a species related to S. colpophylla, is based on female heterogamety, a sex determination system that is unique among the Silene species studied to date. Our phylogenetic data support the placing of S. otites and S. colpophylla in the subsection Otites and the analysis of ancestral states suggests that the most recent common ancestor of S. otites and S. colpophylla was most probably dioecious. These observations imply that a switch from XX/XY sex determination to a ZZ/ZW system (or vice versa) occurred in the subsection Otites. This is the first report of two different types of heterogamety within one plant genus of this mostly nondioecious plant family.     

Dual Gating Mechanism and Function of P2X7 Receptor Channels

The ATP-gated P2X7 receptor channel (P2X7R) operates as a cytolytic and apoptotic receptor but also controls sustained cellular responses, including cell growth and proliferation. However, it has not been clarified how the same receptor mediates such opposing effects. To address this question, we have combined electrophysiological, imaging, and mathematical studies using wild-type and mutant rat P2X7Rs. Activation of naïve (not previously stimulated) receptors by low agonist concentrations caused monophasic slow desensitizing currents and internalization of receptors without other changes in the cellular morphology, much like other P2XRs. In contrast, saturating agonist concentrations induced high-amplitude biphasic currents, reflecting pore dilation and causing rapid cell swelling and lysis. The existence of these two signaling patterns was accounted for using a revised Markov-state model that included, in addition to naïve and sensitized states, desensitized states. Occupancy of one or two ATP-binding sites of naïve receptors favored a slow transition to desensitized states, whereas occupancy of the third binding site favored a transition to sensitized/dilated states. Consistent with model predictions, nondilating P2X7R mutants always generated desensitizing currents. These results suggest that the level of saturation of the ligand binding sites determines the nature of the P2X7R gating and cellular actions.     

Vhc1, a novel transporter belonging to the family of electroneutral cation–Cl− cotransporters,participates in the regulation of cation content and morphology of Saccharomyces cerevisiae vacuoles

Cation–Cl^? cotransporters (CCCs) are integral membrane proteins which catalyze the coordinated symport of Cl^? with Na⁺ and/or K⁺ ions in plant and mammalian cells. Here we describe the first Saccharomyces cerevisiae CCC protein, encoded by the YBR235w open reading frame. Subcellular localization studies showed that this yeast CCC is targeted to the vacuolar membrane. Deletion of the YBR235w gene in a salt-sensitive strain (lacking the plasma-membrane cation exporters) resulted in an increased sensitivity to high KCl, altered vacuolar morphology control and decreased survival upon hyperosmotic shock. In addition, deletion of the YBR235w gene in a mutant strain deficient in K⁺ uptake produced a significant growth advantage over the parental strain under K⁺-limiting conditions, and a hypersensitivity to the exogenous K⁺/H⁺ exchanger nigericin. These results strongly suggest that we have identified a novel yeast vacuolar ion transporter mediating a K⁺–Cl^? cotransport and playing a role in vacuolar osmoregulation. Considering its identified function, we propose to refer to the yeast YBR235w gene as VHC1 (vacuolar protein homologous to CCC family 1).     

The first case of fatal pneumonia caused by Panton–Valentine leukocidin-producing Staphylococcus aureus in an infant in the Czech Republic

Panton–Valentine leukocidin-producing strains of Staphylococcus aureus can cause severe skin and soft tissue infections and necrotizing pneumonia with a high mortality rate. This is a report on the first case of fatal pneumonia with mediastinitis in an infant in the Czech Republic. The causative agent was a methicillin-susceptible S. aureus strain with pronounced production of the PVL toxin and hyperproduction of enterotoxin A.     

Pepsin Digest of Wheat Gliadin Fraction Increases Production of IL-1β via TLR4/MyD88/TRIF/MAPK/NF-κB Signaling Pathway and an NLRP3 Inflammasome Activation

Celiac disease (CD) is a gluten-responsive, chronic inflammatory enteropathy. IL-1 cytokine family members IL-1β and IL-18 have been associated with the inflammatory conditions in CD patients. However, the mechanisms of IL-1 molecule activation in CD have not yet been elucidated. We show in this study that peripheral blood mononuclear cells (PBMC) and monocytes from celiac patients responded to pepsin digest of wheat gliadin fraction (PDWGF) by a robust secretion of IL-1β and IL-1α and a slightly elevated production of IL-18. The analysis of the upstream mechanisms underlying PDWGF-induced IL-1β production in celiac PBMC show that PDWGF-induced de novo pro-IL-1β synthesis, followed by a caspase-1 dependent processing and the secretion of mature IL-1β. This was promoted by K+ efflux and oxidative stress, and was independent of P2X7 receptor signaling. The PDWGF-induced IL-1β release was dependent on Nod-like receptor family containing pyrin domain 3 (NLRP3) and apoptosis-associated speck like protein (ASC) as shown by stimulation of bone marrow derived dendritic cells (BMDC) from NLRP3^−/− and ASC^−/− knockout mice. Moreover, treatment of human PBMC as well as MyD88^−/− and Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-β (TRIF)^−/− BMDC illustrated that prior to the activation of caspase-1, the PDWGF-triggered signal constitutes the activation of the MyD88/TRIF/MAPK/NF-κB pathway. Moreover, our results indicate that the combined action of TLR2 and TLR4 may be required for optimal induction of IL-1β in response to PDWGF. Thus, innate immune pathways, such as TLR2/4/MyD88/TRIF/MAPK/NF-κB and an NLRP3 inflammasome activation are involved in wheat proteins signaling and may play an important role in the pathogenesis of CD.     

ZrFsy1, a High-Affinity Fructose/H+ Symporter from Fructophilic Yeast Zygosaccharomyces rouxii

Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H⁺ symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H⁺ symporter, with K_m 0.45±0.07 mM and V_max 0.57±0.02 mmol h⁻¹ (gdw) ⁻¹. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2%) and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H⁺ symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system.     

Staphylococcus aureus Sortase A-Mediated Incorporation of Peptides: Effect of Peptide Modification on Incorporation

The endogenous Staphylococcus aureus sortase A (SrtA) transpeptidase covalently anchors cell wall-anchored (CWA) proteins equipped with a specific recognition motif (LPXTG) into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITC)LPETG-amide) into the bacterial cell wall, albeit at high concentrations of 500 μM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i) by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 μM, 100 μM and 250 μM; (ii) Substituting aspartic acid (E) for methionine increased the incorporation of the resulting K(FITC)LPMTG-amide approximately three times at all concentrations tested; (iii) conjugation of the lipid II binding antibiotic vancomycin to K(FITC)LPMTG-amide resulted in the same incorporation levels as K(FITC)LPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall.     

Differential Effects of apoE4 and Activation of ABCA1 on Brain and Plasma Lipoproteins

Apolipoprotein E4 (apoE4), the leading genetic risk factor for Alzheimer''s disease (AD), is less lipidated compared to the most common and AD-benign allele, apoE3. We have recently shown that i.p. injections of the ATP-binding cassette A1 (ABCA1) agonist peptide CS-6253 to apoE mice reverse the hypolipidation of apoE4 and the associated brain pathology and behavioral deficits. While in the brain apoE is the main cholesterol transporter, in the periphery apoE and apoA-I both serve as the major cholesterol transporters. We presently investigated the extent to which apoE genotype and CS-6253 treatment to apoE3 and apoE4-targeted replacement mice affects the plasma levels and lipid particle distribution of apoE, and those of plasma and brain apoA-I and apoJ. This revealed that plasma levels of apoE4 were lower and eluted faster following FPLC than plasma apoE3. Treatment with CS-6253 increased the levels of plasma apoE4 and rendered the elution profile of apoE4 similar to that of apoE3. Similarly, the levels of plasma apoA-I were lower in the apoE4 mice compared to apoE3 mice, and this effect was partially reversed by CS-6253. Conversely, the levels of apoA-I in the brain which were higher in the apoE4 mice, were unaffected by CS-6253. The plasma levels of apoJ were higher in apoE4 mice than apoE3 mice and this effect was abolished by CS-6253. Similar but less pronounced effects were obtained in the brain. In conclusion, these results suggest that apoE4 affects the levels of apoA-I and apoJ and that the anti-apoE4 beneficial effects of CS-6253 may be related to both central and peripheral mechanisms.     

Ontogenetic integration between force production and force reception: a case study in Ctenomys (Rodentia: Caviomorpha)

During ontogeny, complex adaptations undergo changes that sometimes entail different functional capabilities. This fact constrains the behaviour of organisms at each developmental stage. Rodents have ever‐growing incisors for gnawing, and a powerful jaw musculature. The incisors are long enough, relative to their diameter, to be affected by bending stresses. This is particularly true in the subterranean Ctenomys that uses its incisors for digging. We measured bite force (BF) in individuals of different ages using a force transducer. We estimated incisor section modulus Z, a geometrical parameter proportional to bending strength. A relative strength indicator was calculated as S = Z/BF incisor length. We found that ontogenetic BF scales to body mass with positive allometry. However, an anova showed non‐significant differences in S, neither between sexes nor among age classes. This result implies that during growth, incisors might have a rather similar ability to withstand bending stresses from increasing masticatory forces, what may be considered evidence of ontogenetic integration of force production (by muscles) and force reception (by the incisors). This fact well correlates with the observation that pups and juveniles of C. talarum incorporate solid foods shortly after birth, and they are able to dig burrows early in life.