Neobiosynthesis of Glycosphingolipids by Plasma Membrane-associated Glycosyltransferases

Gangliosides, complex glycosphingolipids containing sialic acids, are synthesized in the endoplasmic reticulum and in the Golgi complex. These neobiosynthesized gangliosides move via vesicular transport to the plasma membrane, becoming components of the external leaflet. Gangliosides can undergo endocytosis followed by recycling to the cell surface or sorting to the Golgi complex or lysosomes for remodeling and catabolism. Recently, glycosphingolipid catabolic enzymes (glycohydrolases) have been found to be associated with the plasma membrane, where they display activity on the membrane components. In this work, we demonstrated that ecto-ganglioside glycosyltransferases may catalyze ganglioside synthesis outside the Golgi compartment, particularly at the cell surface. Specifically, we report the first direct evidence of expression and activity of CMP-NeuAc:GM3 sialyltransferase (Sial-T2) at the cell surface of epithelial and melanoma cells, with membrane-integrated ecto-Sial-T2 being able to sialylate endogenously synthesized GM3 ganglioside as well as exogenously incorporated substrate. Interestingly, we also showed that ecto-Sial-T2 was able to synthesize GD3 ganglioside at the cell surface using the endogenously synthesized cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) available at the extracellular milieu. In addition, the expression of UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase (GalNAc-T) was also detected at the cell surface of epithelial cells, whose catalytic activity was only observed after feeding the cells with exogenous GM3 substrate. Thus, the relative interplay between the plasma membrane-associated glycosyltransferase and glycohydrolase activities, even when acting on a common substrate, emerges as a potential level of regulation of the local glycosphingolipid composition in response to different external and internal stimuli.     

Pulmonary arterial endothelial cells affect the redox status of coenzyme Q0

The pulmonary endothelium is capable of reducing certain redox-active compounds as they pass from the systemic venous to the arterial circulation. This may have important consequences with regard to the pulmonary and systemic disposition and biochemistry of these compounds. Because quinones comprise an important class of redox-active compounds with a range of physiological, toxicological, and pharmacological activities, the objective of the present study was to determine the fate of a model quinone, coenzyme Q0 (Q), added to the extracellular medium surrounding pulmonary arterial endothelial cells in culture, with particular attention to the effect of the cells on the redox status of Q in the medium. Spectrophotometry, electron paramagnetic resonance (EPR), and high-performance liquid chromatography (HPLC) demonstrated that, when the oxidized form Q is added to the medium surrounding the cells, it is rapidly converted to its quinol form (QH2) with a small concentration of semiquinone (Q*-) also detectable. The isolation of cell plasma membrane proteins revealed an NADH-Q oxidoreductase located on the outer plasma membrane surface, which apparently participates in the reduction process. In addition, once formed the QH2 undergoes a cyanide-sensitive oxidation by the cells. Thus, the actual rate of Q reduction by the cells is greater than the net QH2 output from the cells.     

Duroquinone reduction during passage through the pulmonary circulation

The lungs can substantially influence the redox status of redox-active plasma constituents. Our objective was to examine aspects of the kinetics and mechanisms that determine pulmonary disposition of redox-active compounds during passage through the pulmonary circulation. Experiments were carried out on rat and mouse lungs with 2,3,5,6-tetramethyl-1,4-benzoquinone [duroquinone (DQ)] as a model amphipathic quinone reductase substrate. We measured DQ and durohydroquinone (DQH2) concentrations in the lung venous effluent after injecting, or while infusing, DQ or DQH2 into the pulmonary arterial inflow. The maximum net rates of DQ reduction to DQH2 in the rat and mouse lungs were approximately 4.9 and 2.5 micromol. min(-1).g dry lung wt(-1), respectively. The net rate was apparently the result of freely permeating access of DQ and DQH2 to tissue sites of redox reactions, dominated by dicumarol-sensitive DQ reduction to DQH2 and cyanide-sensitive DQH2 reoxidation back to DQ. The dicumarol sensitivity along with immunodetectable expression of NAD(P)H-quinone oxidoreductase 1 (NQO1) in the rat lung tissue suggest cytoplasmic NQO1 as the dominant site of DQ reduction. The effect of cyanide on DQH2 oxidation suggests that the dominant site of oxidation is complex III of the mitochondrial electron transport chain. If one envisions DQ as a model compound for examining the disposition of amphipathic NQO1 substrates in the lungs, the results are consistent with a role for lung NQO1 in determining the redox status of such compounds in the circulation. For DQ, the effect is conversion of a redox-cycling, oxygen-activating quinone into a stable hydroquinone.     

Pathogen-driven selection and worldwide HLA class I diversity

The human leukocyte antigen (HLA; known as MHC in other vertebrates) plays a central role in the recognition and presentation of antigens to the immune system and represents the most polymorphic gene cluster in the human genome [1]. Pathogen-driven balancing selection (PDBS) has been previously hypothesized to explain the remarkable polymorphism in the HLA complex, but there is, as yet, no direct support for this hypothesis [2 and 3]. A straightforward prediction coming out of the PDBS hypothesis is that populations from areas with high pathogen diversity should have increased HLA diversity in relation to their average genomic diversity. We tested this prediction by using HLA class I genetic diversity from 61 human populations. Our results show that human colonization history explains a substantial proportion of HLA genetic diversity worldwide. However, between-population variation at the HLA class I genes is also positively correlated with local pathogen richness (notably for the HLA B gene), thus providing support for the PDBS hypothesis. The proportion of variations explained by pathogen richness is higher for the HLA B gene than for the HLA A and HLA C genes. This is in good agreement with both previous immunological and genetic data suggesting that HLA B could be under a higher selective pressure from pathogens.     

Heterogeneous vancomycin-intermediate susceptibility in a community-associated methicillin-resistant Staphylococcus aureus epidemic clone, in a case of Infective Endocarditis in Argentina

Background

There has been considerable effort to discover plant-derived antibacterials against methicillin-resistant strains of Staphylococcus aureus (MRSA) which have developed resistance to most existing antibiotics, including the last line of defence, vancomycin. Pentacyclic triterpenoid, a biologically diverse plant-derived natural product, has been reported to show anti-staphylococcal activities. The objective of this study is to evaluate the interaction between three pentacyclic triterpenoid and standard antibiotics (methicillin and vancomycin) against reference strains of Staphylococcus aureus.

Methods and Results

The activity of the standard antibiotics and compounds on reference methicillin-sensitive and resistant strains of S. aureus were determined using the macrodilution broth method. The minimum inhibitory concentration (MIC) of the compounds was compared with that of the standard antibiotics. The interaction between any two antimicrobial agents was estimated by calculating the fractional inhibitory concentration (FIC index) of the combination. The various combinations of antibiotics and compounds reduced the MIC to a range of 0.05 to 50%.

Conclusion

Pentacyclic triterpenoids have shown anti-staphylococcal activities and although individually weaker than common antibiotics produced from bacteria and fungi, synergistically these compounds may use different mechanism of action or pathways to exert their antimicrobial effects, as implicated in the lowered MICs. Therefore, the use of current antibiotics could be maintained in their combination with plant-derived antibacterial agents as a therapeutic option in the treatment of S. aureus infections.     

Establishment and characterization of atypical fibroblasts from human adult liver contributing to hepatocyte cord-like arrangement

We report the phenotypic and functional characterization of fibroblasts established in culture from the non-parenchymal epithelial cell populations of adult human livers. Human liver fibroblasts (hLF) expressed mesenchymal antigens vimentin, alpha-smooth muscle actin, collagen, fibronectin, CD73, CD90, CD105, and CD166 together with non-mesenchymal antigens cytokeratins 8 and 18, glial fibrillary acidic protein, and nestin. Mixed cell lineage-specific protein expression was not associated with stem-like cell properties. Coculturing hepatocytes onto confluent hLF showed that they survived and maintained metabolic activity such as albumin, glycogen, and urea production. Moreover, hepatocytes formed cord-like arrangements resembling those established in vivo. Hepatocyte arrangement depended on cell-to-cell contact and the tissue origin of fibroblasts. Time-lapse video imaging of cocultured cells showed that hepatocyte arrangement was coordinated by the stretching and shortening of underneath hLF. Our data suggest that hLF may represent resident fibroblasts of the adult human liver, which could assume guiding functions for hepatic epithelial cells.     

Genomic data reveal deep genetic structure but no support for current taxonomic designation in a grasshopper species complex

Taxonomy has traditionally relied on morphological and ecological traits to interpret and classify biological diversity. Over the last decade, technological advances and conceptual developments in the field of molecular ecology and systematics have eased the generation of genomic data and changed the paradigm of biodiversity analysis. Here we illustrate how traditional taxonomy has led to species designations that are supported neither by high throughput sequencing data nor by the quantitative integration of genomic information with other sources of evidence. Specifically, we focus on Omocestus antigai and Omocestus navasi, two montane grasshoppers from the Pyrenean region that were originally described based on quantitative phenotypic differences and distinct habitat associations (alpine vs. Mediterranean‐montane habitats). To validate current taxonomic designations, test species boundaries, and understand the factors that have contributed to genetic divergence, we obtained phenotypic (geometric morphometrics) and genome‐wide SNP data (ddRADSeq) from populations covering the entire known distribution of the two taxa. Coalescent‐based phylogenetic reconstructions, integrative Bayesian model‐based species delimitation, and landscape genetic analyses revealed that populations assigned to the two taxa show a spatial distribution of genetic variation that do not match with current taxonomic designations and is incompatible with ecological/environmental speciation. Our results support little phenotypic variation among populations and a marked genetic structure that is mostly explained by geographic distances and limited population connectivity across the abrupt landscapes characterizing the study region. Overall, this study highlights the importance of integrative approaches to identify taxonomic units and elucidate the evolutionary history of species.     

How Much Does Ultraviolet Radiation Contribute to the Feeding Performance of Rainbow Trout, Oncorhynchus mykiss, Juveniles under Natural Illumination?

Most research on environmental effects of ultraviolet radiation (UVR) has focused on its potential negative consequences. However, natural UVR can also be beneficial to living organisms (e.g., vitamin D synthesis, UV vision, germicide activity). UV vision has been demonstrated in a variety of animals including several invertebrates and vertebrates. Juvenile rainbow trout, Oncorhynchus mykiss, has a retinal photoreceptor, which is sensitive to UVR between 360 and 370 nm. Among other functions, UV vision has been proposed to contribute to prey detection by enhancing the contrast between the prey and its background. We performed a series of feeding experiments with juvenile rainbow trout and several zooplankters as prey. The fish were allowed to feed either under full solar radiation, or under solar radiation from which the UV component had been removed using a long-pass cut off filter. We found that the removal of UV wavelengths had no effect on the number of prey eaten or on the preference for particular food items. This is contrary to published studies reporting prey detection enhancement mediated by UV vision in rainbow trout. This disparity in the results may be due to our use of natural radiation instead of artificial UV sources, in which the visible component is poorly represented. Although our results do not disproof the presence of UV vision in juvenile rainbow trout, they do cast doubts about its significance in enhancing feeding performance in a natural light environment.     

Alteration of Plasma Membrane Organization by an Anticancer Lysophosphatidylcholine Analogue Induces Intracellular Acidification and Internalization of Plasma Membrane Transporters in Yeast

The lysophosphatidylcholine analogue edelfosine is a potent antitumor lipid that targets cellular membranes. The underlying mechanisms leading to cell death remain controversial, although two cellular membranes have emerged as primary targets of edelfosine, the plasma membrane (PM) and the endoplasmic reticulum. In an effort to identify conditions that enhance or prevent the cytotoxic effect of edelfosine, we have conducted genome-wide surveys of edelfosine sensitivity and resistance in Saccharomyces cerevisiae presented in this work and the accompanying paper (Cuesta-Marbán, Á., Botet, J., Czyz, O., Cacharro, L. M., Gajate, C., Hornillos, V., Delgado, J., Zhang, H., Amat-Guerri, F., Acuña, A. U., McMaster, C. R., Revuelta, J. L., Zaremberg, V., and Mollinedo, F. (January 23, 2013) J. Biol. Chem. 288,), respectively. Our results point to maintenance of pH homeostasis as a major player in modulating susceptibility to edelfosine with the PM proton pump Pma1p playing a main role. We demonstrate that edelfosine alters PM organization and induces intracellular acidification. Significantly, we show that edelfosine selectively reduces lateral segregation of PM proteins like Pma1p and nutrient H⁺-symporters inducing their ubiquitination and internalization. The biology associated to the mode of action of edelfosine we have unveiled includes selective modification of lipid raft integrity altering pH homeostasis, which in turn regulates cell growth.     

Staphylococcus aureus Mutants Lacking the LytR-CpsA-Psr Family of Enzymes Release Cell Wall Teichoic Acids into the Extracellular Medium

The LytR-CpsA-Psr (LCP) proteins are thought to transfer bactoprenol-linked biosynthetic intermediates of wall teichoic acid (WTA) to the peptidoglycan of Gram-positive bacteria. In Bacillus subtilis, mutants lacking all three LCP enzymes do not deposit WTA in the envelope, while Staphylococcus aureus Δlcp mutants display impaired growth and reduced levels of envelope phosphate. We show here that the S. aureus Δlcp mutant synthesized WTA yet released ribitol phosphate polymers into the extracellular medium. Further, Δlcp mutant staphylococci no longer restricted the deposition of LysM-type murein hydrolases to cell division sites, which was associated with defects in cell shape and increased autolysis. Mutations in S. aureus WTA synthesis genes (tagB, tarF, or tarJ2) inhibit growth, which is attributed to the depletion of bactoprenol, an essential component of peptidoglycan synthesis (lipid II). The growth defect of S. aureus tagB and tarFJ mutants was alleviated by inhibition of WTA synthesis with tunicamycin, whereas the growth defect of the Δlcp mutant was not relieved by tunicamycin treatment or by mutation of tagO, whose product catalyzes the first committed step of WTA synthesis. Further, sortase A-mediated anchoring of proteins to peptidoglycan, which also involves bactoprenol and lipid II, was not impaired in the Δlcp mutant. We propose a model whereby the S. aureus Δlcp mutant, defective in tethering WTA to the cell wall, cleaves WTA synthesis intermediates, releasing ribitol phosphate into the medium and recycling bactoprenol for peptidoglycan synthesis.