Fluorescence correlation studies of lipid domains in model membranes

Advances in optical microscopy techniques and single-molecule detection have paved the way to exploring new approaches for investigating membrane dynamics and organization, thereby revealing details on the processing of signals, complex association/dissociation, chemical reactions and transport at and around the membrane. These events rely on a tight regulation of lipid-protein and protein-protein interactions in space and time. Fluorescence Correlation Spectroscopy (FCS) provides exquisite sensitivity in measuring local concentrations, association/dissociation constants, chemical rate constants and, in general, in probing the chemical environment of the species of interest and its interactions with potential partners. Here, we review some applications of FCS to lipid and protein organization in biomimetic membranes with lateral heterogeneities, which share some physico-chemical properties with cellular rafts. What we learn from investigations of lipid-lipid and lipid-protein interactions in simple model membranes can be regarded as an essential basic lecture for studies in more complex cellular membranes.     

Effects of platelets on the protein expression in aortic segments: A proteomic approach

It is well known the effects of the vascular wall on platelet activity but little is known about the effects of platelets on the proteins expression in the vascular wall. We analyzed whether platelets may modify the protein expression in the vascular wall. We used an in vitro model coincubating human platelet rich plasma (PRP) with control and 10 ng/ml tumor necrosis factor‐α (TNF‐α)‐preincubated bovine aortic segments. 2DE, mass spectrometry and Western blot analysis were used to determine changes in the expression of proteins associated with the cytoskeleton and energetic metabolism in the aortic segments. In control healthy vascular wall, only the cytoskeleton‐related proteins expression was modified by PRP. However, when PRP was coincubated with TNF‐α pre‐stimulated aortic segments lesser number of cytoskeleton‐related proteins were modified. With respect to energetic metabolism, in control segments, PRP failed to modify any of the analyzed energetic‐related proteins. However, in TNF‐α‐preincubated segments the presence of PRP upexpressed glyceraldehyde‐3‐phosphate dehydrogenase. Moreover, by western blot experiments it was observed that in TNF‐α‐preincubated segments the expression of fructose 1,6‐bisphosphate aldolase was downregulated by platelets. However, no differences were found in the expression of triosephosphate isomerase and ATP synthase α‐chain. In addition, the activity of fructose 1,6‐bisphosphate aldolase and piruvate content was significantly reduced without modification on triosephosphate isomerase activity. In conclusion, the crosstalk between platelets and vascular wall is bidirectional and platelets regulated in the vascular wall the expression of proteins associated with the cytoskeleton and energetic metabolism, particularly in the healthy vascular wall. J. Cell. Biochem. 111: 889–898, 2010. © 2010 Wiley‐Liss, Inc.     

Irradiation with heavy-ion particles changes the cellular distribution of human histone acetyltransferase HAT1

Hat1 was the first histone acetyltransferase identified; however, its biological function is still unclear. In this report, it is shown for the first time that human Hat1 has two isoforms. Isoform a has 418 amino acids (aa) and is localized exclusively in the nuclear matrix of normal human keratinocytes (NHKs). Isoform b has 334 aa and is located in the cytoplasm, the nucleoplasm, attached to the chromatin and to the nuclear matrix. Immunohistochemical analyses revealed that the bulk of Hat1 is confined to the nucleus, with much lesser amounts in the cytoplasm. Cells undergoing mitotic division have an elevated amount of Hat1 compared to those that are non-mitotic. Senescent cells, however, exhibit a higher concentration of Hat1 in the cytoplasm compare to proliferating cells and the amount of Hat1 in the nucleus decreases with the progression of senescence. NHKs exposed to hydrogen peroxide (H₂O₂) or to a beam of high mass and energy ion particles displayed bright nuclear staining for Hat1, a phenotype that was not observed in NHKs exposed to γ-rays. We established that the enhanced nuclear staining for Hat1 in response to these treatments is regulated by the PI3K and the mitogen-activated protein kinase signaling pathways. Our observations clearly implicate Hat1 in the cellular response assuring the survival of the treated cells.     

Inositol catabolism, a key pathway in sinorhizobium meliloti for competitive host nodulation

The nitrogen-fixing symbiont of alfalfa, Sinorhizobium meliloti, is able to use myo-inositol as the sole carbon source. Putative inositol catabolism genes (iolA and iolRCDEB) have been identified in the S. meliloti genome based on their similarities with the Bacillus subtilis iol genes. In this study, functional mutational analysis revealed that the iolA and iolCDEB genes are required for growth not only with the myo-isomer but also for growth with scyllo- and d-chiro-inositol as the sole carbon source. An additional, hypothetical dehydrogenase of the IdhA/MocA/GFO family encoded by the smc01163 gene was found to be essential for growth with scyllo-inositol, whereas the idhA-encoded myo-inositol dehydrogenase was responsible for the oxidation of d-chiro-inositol. The putative regulatory iolR gene, located upstream of iolCDEB, encodes a repressor of the iol genes, negatively regulating the activity of the myo- and the scyllo-inositol dehydrogenases. Mutants with insertions in the iolA, smc01163, and individual iolRCDE genes could not compete against the wild type in a nodule occupancy assay on alfalfa plants. Thus, a functional inositol catabolic pathway and its proper regulation are important nutritional or signaling factors in the S. meliloti-alfalfa symbiosis.     

All-or-none versus graded: single-vesicle analysis reveals lipid composition effects on membrane permeabilization

We report a single-vesicle approach to compare the all-or-none and graded mechanisms of lipid bilayer permeabilization by CpreTM and NpreTM, two peptides derived from the membrane-proximal external region of the HIV fusion glycoprotein gp41subunit. According to bulk requenching assays, these peptides permeabilize large unilamellar vesicles via all-or-none and graded mechanisms, respectively. Visualization of the process using giant unilamellar vesicles shows that the permeabilization of individual liposomes by these two peptides differs in kinetics, degree of dye filling, and stability of the permeabilized state. All-or-none permeabilization by CpreTM is characterized by fast and total filling of the individual vesicles. This process is usually accompanied by the formation of stably open pores, as judged from the capacity of the vesicles to incorporate a second dye added after several hours. In contrast, graded permeabilization by NpreTM is transient and exhibits slower kinetics, which leads to partial filling of the individual liposomes. Of importance, quantitative analysis of vesicle population distribution allowed the identification of mixed mechanisms of membrane permeabilization and the assessment of cholesterol effects. Specifically, the presence of this viral envelope lipid increased the stability of the permeating structures, which may have implications for the fusogenic activity of gp41.     

Neutral glycosphingolipids in human blood: a precise mass spectrometry analysis with special reference to lipoprotein-associated Shiga toxin receptors

Shiga toxin (Stx)-producing Escherichia coli are the leading cause of hemorrhagic colitis and life-threatening extraintestinal complications in humans. Stx1 and Stx2 are transferred by yet to be delineated mechanisms from the intestine to the circulation where they injure microvascular endothelial cells. The resulting vascular lesions cause renal failure and brain damage. Because lipoproteins are potential carriers of Stx through the circulation, we investigated human lipoprotein-associated neutral glycosphingolipids (GSLs) with emphasis on high (globotriaosylceramide) and low (globotetraosylceramide) affinity Stx-receptors. TLC overlay employing Stx1, Stx2, and anti-GSL antibodies demonstrated preferential distribution of globo-series GSLs to very low- and low-density lipoproteins compared with minor association with high-density lipoproteins. Electrospray ionization quadrupole time-of-flight mass spectrometry portrayed C24:0/C24:1 and C16:0 as the major fatty acid of the ceramide moieties of Stx-receptors carrying nonvarying d18:1 sphingosine. This structural heterogeneity was also found in precursor lactosylceramide, glucosylceramide, and galactosylceramide, the last showing an exceptionally high degree of hydroxylated C24 fatty acids. Our findings provide the basis for exploring the functional role of lipoprotein-associated Stx-receptors in human blood.     

Spontaneous succession in limestone quarries as an effective restoration tool for endangered arthropods and plants

1. The view of post-mining sites is rapidly changing among ecologists and conservationists, as sensitive restoration using spontaneous succession may turn such sites into biodiversity refuges in human-exploited regions. However, technical reclamation, consisting of covering the sites by topsoil, sowing fast-growing herb mixtures and planting trees, is still commonly adopted. Until now, no multi-taxa study has compared technically reclaimed sites and sites left with spontaneous succession.
2. We sampled communities of vascular plants and 10 arthropod groups in technically reclaimed and spontaneously restored plots in limestone quarries in the Bohemian Karst, Czech Republic. For comparison, we used paired t -tests and multivariate methods, emphasizing red-list status and habitat specialization of individual species.
3. We recorded 692 species of target taxa, with a high proportion of red-listed (10%) and xeric specialist (14%) species, corroborating the great conservation potential of the quarries.
4. Spontaneously restored post-mining sites did not differ in species richness from the technical reclaimed sites but they supported more rare species. The microhabitat cover of leaf litter, herbs and moss, were all directly influenced by the addition of topsoil during reclamation.
5. Synthesis and applications. Our results show that the high conservation potential of limestone quarries could be realized by allowing succession to progress spontaneously with minimal intervention. Given the threat to semi-natural sparsely vegetated habitats in many regions, active restoration measures at post-mining sites should be limited to maintenance of early successional stages, instead of acceleration of succession.