Ceramide: physiological and pathophysiological aspects

Ceramide generated in the cell membrane has been shown to be central for the induction of apoptosis by death receptors and many stress stimuli such as gamma-irradiation, UV-light or infection with pathogens. Ceramide reorganizes cell membranes and forms large ceramide-enriched membrane domains that serve the spatial and temporal organization of the cellular signalosome upon activation. Thus, ceramide-enriched membrane domains mediate clustering of CD95 and DR5 to facilitate apoptosis, and they are also critically involved in apoptosis after irradiation, UV-light and infection with Pseudomonas aeruginosa. Since ceramide-enriched membrane domains amplify signals, their function is not restricted to the induction of apoptosis and it was shown that ceramide-enriched membrane domains are also involved in internalization of pathogens and the control of cytokine release from infected epithelial cells. Recent studies support the notion that changes of the ceramide metabolism are also critically involved in human diseases, for instance neurological disorders, cancer, infectious diseases and Wilson's disease.     

Indole-3-acetaldoxime-derived compounds restrict root colonization in the beneficial interaction between Arabidopsis roots and the endophyte Piriformospora indica

The growth-promoting and root-colonizing endophyte Piriformospora indica induces camalexin and the expression of CYP79B2, CYP79B3, CYP71A13, PAD3, and WRKY33 required for the synthesis of indole-3-acetaldoxime (IAOx)-derived compounds in the roots of Arabidopsis seedlings. Upregulation of the mRNA levels by P. indica requires cytoplasmic calcium elevation and mitogen-activated protein kinase 3 but not root-hair-deficient 2, radical oxygen production, or the 3-phosphoinositide-dependent kinase 1/oxidative signal-inducible 1 pathway. Because P. indica-mediated growth promotion is impaired in cyp79B2 cyp79B3 seedlings, while pad3 seedlings-which do not accumulate camalexin-still respond to the fungus, IAOx-derived compounds other than camalexin (e.g., indole glucosinolates) are required during early phases of the beneficial interaction. The roots of cyp79B2 cyp79B3 seedlings are more colonized than wild-type roots, and upregulation of the defense genes pathogenesis-related (PR)-1, PR-3, PDF1.2, phenylalanine ammonia lyase, and germin indicates that the mutant responds to the lack of IAOx-derived compounds by activating other defense processes. After 6 weeks on soil, defense genes are no longer upregulated in wild-type, cyp79B2 cyp79B3, and pad3 roots. This results in uncontrolled fungal growth in the mutant roots and reduced performance of the mutants. We propose that a long-term harmony between the two symbionts requires restriction of root colonization by IAOx-derived compounds.     

Conceptual data modelling for bioinformatics

Current research in the biosciences depends heavily on the effective exploitation of huge amounts of data. These are in disparate formats, remotely dispersed, and based on the different vocabularies of various disciplines. Furthermore, data are often stored or distributed using formats that leave implicit many important features relating to the structure and semantics of the data. Conceptual data modelling involves the development of implementation-independent models that capture and make explicit the principal structural properties of data. Entities such as a biopolymer or a reaction, and their relations, eg catalyses, can be formalised using a conceptual data model. Conceptual models are implementation-independent and can be transformed in systematic ways for implementation using different platforms, eg traditional database management systems. This paper describes the basics of the most widely used conceptual modelling notations, the ER (entity-relationship) model and the class diagrams of the UML (unified modelling language), and illustrates their use through several examples from bioinformatics. In particular, models are presented for protein structures and motifs, and for genomic sequences.     

Improved high-performance liquid chromatographic separation of peptidoglycan isolated from various Staphylococcus aureus strains for mass spectrometric characterization

Reversed-phase high-performance liquid chromatography (RP-HPLC) of muropeptides, obtained by muramidase digestion of peptidoglycan in combination with amino acid analysis and plasma desorption time-of-flight mass spectrometry is today by far the best tool to analyze the fine structure of the peptidoglycans. Here we report further improvements of the RP-HPLC separation of muropeptides for analyzing the peptidoglycans of various methicillin-resistant strains of Staphylococcus aureus, with emphasis on a more detailed characterization of the interpeptide bridge of the peptidoglycans of this species.     

Endothelial Progenitor Cell Function,Apoptosis, and Telomere Length in Overweight/Obese Humans

Excess adiposity is associated with increased cardiovascular morbidity and mortality. Endothelial progenitor cells (EPCs) play an important role in vascular repair. We tested the hypothesis that increased adiposity is associated with EPC dysfunction, characterized by diminished capacity to release angiogenic cytokines, increased apoptotic susceptibility, reduced cell migration, and shorter telomere length. A total of 67 middle‐aged and older adults (42–67 years) were studied: 25 normal weight (normal weight; BMI: 18.5–24.9 kg/m²) and 42 overweight/obese (overweight/obese; BMI: 25.0–34.9 kg/m²). Cells with phenotypic EPC characteristics were isolated from peripheral blood. EPC release of vascular endothelial growth factor (VEGF) and granulocyte colony–stimulating factor (G‐CSF) was determined in the absence and presence of phytohemagglutinin (10 µg/ml). Intracellular active caspase‐3 and cytochrome c concentrations were determined by immunoassay. Migratory activity of EPCs in response to VEGF (2 ng/ml) and stromal cell–derived factor‐1α (SDF‐1α; 10 ng/ml) was determined by Boyden chamber. Telomere length was assessed by Southern hybridization. Phytohemagglutinin‐stimulated release of VEGF (90.6 ± 7.6 vs. 127.2 ± 11.6 pg/ml) and G‐CSF (896.1 ± 77.4 vs. 1,176.3 ± 126.3 pg/ml) was ～25% lower (P < 0.05) in EPCs from overweight/obese vs. normal weight subjects. Staurosporine induced a ～30% greater (P < 0.05) increase in active caspase‐3 in EPCs from overweight/obese (2.8 ± 0.2 ng/ml) compared with normal weight (2.2 ± 0.2) subjects. There were no significant differences in EPC migration to either VEGF or SDF‐1α. Telomere length did not differ between groups. These results indicate that increased adiposity adversely affects the ability of EPCs to release proangiogenic cytokines and resist apoptosis, potentially compromising their reparative potential.     

Cell volume and the regulation of apoptotic cell death

Apoptosis is a physiological mechanism allowing for the removal of abundant or potentially harmful cells. The hallmarks of apoptosis include degradation of cellular DNA, exposure of phosphatidylserine at the outer leaflet of the cell membrane and cell shrinkage. Phosphatidylserine exposure favours adhesion to macrophages with subsequent phagocytosis of the shrunken apoptotic particles. The interaction of cell volume regulatory mechanisms and apoptosis is illustrated in two different model systems, i.e. (a) lymphocyte apoptosis following stimulation of CD95 receptor and (b) erythrocyte apoptosis upon cell shrinkage. (a) Triggering of CD95 in Jurkat T lymphocytes is paralleled by activation of cell volume regulatory Cl- channels, inhibition of the Na+/H+ exchanger and osmolyte release. The latter coincides with cell shrinkage, DNA fragmentation and phosphatidylserine exposure. CD95 stimulation leads to early inhibition of the voltage gated K+ channel Kv1.3, which may contribute to the inhibition of the Ca2+ release activated Ca2+ channel I(CRAC). (b) Osmotic shock of erythrocytes activates a cell volume regulatory cation conductance allowing the entry not only of Na+ but of Ca2+ as well. Increased cytosolic Ca2+ stimulates a scramblase which disrupts the phosphatidylserine asymmetry of the cell membrane, leading to phosphatidylserine exposure. The cation conductance is further activated by oxidative stress and energy depletion and inhibited by Cl-. Shrinkage of erythrocytes stimulates in addition a sphingomyelinase with subsequent formation of ceramide which potentiates the effect of cytosolic Ca2+ on phosphatidylserine. In conclusion, cell volume-sensitive mechanisms participate in the triggering of apoptosis following receptor stimulation or cell injury.     

Extensive Copy-Number Variation of Young Genes across Stickleback Populations

Duplicate genes emerge as copy-number variations (CNVs) at the population level, and remain copy-number polymorphic until they are fixed or lost. The successful establishment of such structural polymorphisms in the genome plays an important role in evolution by promoting genetic diversity, complexity and innovation. To characterize the early evolutionary stages of duplicate genes and their potential adaptive benefits, we combine comparative genomics with population genomics analyses to evaluate the distribution and impact of CNVs across natural populations of an eco-genomic model, the three-spined stickleback. With whole genome sequences of 66 individuals from populations inhabiting three distinct habitats, we find that CNVs generally occur at low frequencies and are often only found in one of the 11 populations surveyed. A subset of CNVs, however, displays copy-number differentiation between populations, showing elevated within-population frequencies consistent with local adaptation. By comparing teleost genomes to identify lineage-specific genes and duplications in sticklebacks, we highlight rampant gene content differences among individuals in which over 30% of young duplicate genes are CNVs. These CNV genes are evolving rapidly at the molecular level and are enriched with functional categories associated with environmental interactions, depicting the dynamic early copy-number polymorphic stage of genes during population differentiation.