Interactions between gangliosides and proteins in the exoplasmic leaflet of neuronal plasma membranes: A study performed with a tritium-labeled GM1 derivative containing a photoactivable group linked to the oligosaccharide chain

Interactions between gangliosides and proteins at the exoplasmic surface of the sphingolipid-enriched membrane domains can be studied by ganglioside photolabeling combined with cell surface biotin labeling. In the present paper, we report on the results obtained using a novel radioactive photoactivable derivative of GM1 ganglioside, carrying the photoactivable nitrophenylazide group at the external galactose.After cell photolabeling with the radioactive photoactivable derivative of GM1 and cell surface biotin labeling, sphingolipid-enriched domains were prepared from rat cerebellar neurons differentiated in culture and further purified by immunoprecipitation with streptavidin-coupled beads. Among proteins belonging to the sphingolipid-enriched domains that were biotin labeled, thus bearing an exoplasmic domain, a few were also cross-linked by the radioactive photoactivable ganglioside. In particular, two protein bands showing apparent molecular mass of 135 and 35 kDa were intensely photolabeled. The 135 kDa protein was immunologically identified as the GPI-anchored neural cell adhesion molecule TAG-1. These data suggest that hydrophilic interaction between the exoplasmic domains of the protein and the ganglioside sialooligosaccharide chain could exist. Published in 2004.     

Versatile peroxidase oxidation of high redox potential aromatic compounds: site-directed mutagenesis, spectroscopic and crystallographic investigation of three long-range electron transfer pathways

Versatile peroxidases (VP), a recently described family of ligninolytic peroxidases, show a hybrid molecular architecture combining different oxidation sites connected to the heme cofactor. High-resolution crystal structures as well as homology models of VP isoenzymes from the fungus Pleurotus eryngii revealed three possibilities for long-range electron transfer for the oxidation of high redox potential aromatic compounds. The possible pathways would start either at Trp164 or His232 of isoenzyme VPL, and at His82 or Trp170 of isoenzyme VPS1. These residues are exposed, and less than 11 A apart from the heme. With the purpose of investigating their functionality, two single mutations (W164S and H232F) and one double mutation (W164S/P76H) were introduced in VPL that: (i) removed the two pathways in this isoenzyme; and (ii) incorporated the absent putative pathway. Analysis of the variants showed that Trp164 is required for oxidation of two high redox potential model substrates (veratryl alcohol and Reactive Black 5), whereas the two other pathways (starting at His232 and His82) are not involved in long-range electron transfer (LRET). None of the mutations affected Mn2+ oxidation, which would take place at the opposite side of the enzyme. Substitution of Trp164 by His also resulted in an inactive variant, indicating that an indole side-chain is required for activity. It is proposed that substrate oxidation occurs via a protein-based radical. For the first time in a ligninolytic peroxidase such an intermediate species could be detected by low-temperature electron paramagnetic resonance of H2O2-activated VP, and was found to exist at Trp164 as a neutral radical. The H2O2-activated VP was self-reduced in the absence of reducing substrates. Trp164 is also involved in this reaction, which in the W164S variant was blocked at the level of compound II. When analyzing VP crystal structures close to atomic resolution, no hydroxylation of the Trp164 Cbeta atom was observed (even after addition of several equivalents of H2O2). This is in contrast to lignin peroxidase Trp171. Analysis of the crystal structures of both peroxidases showed differences in the environment of the protein radical-forming residue that could affect its reactivity. These variations would also explain differences found for the oxidation of some high redox potential aromatic substrates.     

Identification of immunodominant epitopes of alpha-gliadin in HLA-DQ8 transgenic mice following oral immunization

Celiac disease, triggered by wheat gliadin and related prolamins from barley and rye, is characterized by a strong association with HLA-DQ2 and HLA-DQ8 genes. Gliadin is a mixture of many proteins that makes difficult the identification of major immunodominant epitopes. To address this issue, we expressed in Escherichia coli a recombinant alpha-gliadin (r-alpha-gliadin) showing the most conserved sequence among the fraction of alpha-gliadins. HLA-DQ8 mice, on a gluten-free diet, were intragastrically immunized with a chymotryptic digest of r-alpha-gliadin along with cholera toxin as adjuvant. Spleen and mesenteric lymph node T cell responses were analyzed for in vitro proliferative assay using a panel of synthetic peptides encompassing the entire sequence of r-alpha-gliadin. Two immunodominant epitopes corresponding to peptide p13 (aa 120-139) and p23 (aa 220-239) were identified. The response was restricted to DQ and mediated by CD4+ T cells. In vitro tissue transglutaminase deamidation of both peptides did not increase the response; furthermore, tissue transglutaminase catalyzed extensive deamidation in vitro along the entire r-alpha-gliadin molecule, but failed to elicit new immunogenic determinants. Surprisingly, the analysis of the cytokine profile showed that both deamidated and native peptides induced preferentially IFN-gamma secretion, despite the use of cholera toxin, a mucosal adjuvant that normally induces a Th2 response to bystander Ags. Taken together, these data suggest that, in this model of gluten hypersensitivity, deamidation is not a prerequisite for the initiation of gluten responses.     

Generation of a new class of hNK(2) receptor ligands using the 'fragment approach'

The so called 'fragment approach' was applied in the search for new leads as selective hNK(2) antagonists. A first round of structural space exploration through the use of bond rigidity as scaffold to support the fragments, afforded 27a as 200 nM hNK(2) ligand. Further refinement gave MEN 14933 as a 16 nM hNK(2) ligand, selective versus hNK(1), of a novel class. Conformational analysis was used to study results and plan future work.     

Ultrastructure of testicular macrophages in aging mice

Testicular macrophages of aging mice were studied by TEM. Testicular macrophages retained with Leydig cells the close morphological relationships observed in the adult young animals, but digitations were not found. Lipofuscin granules like those of the Leydig cells from aging mice were observed in the cytoplasm. These organelles were generally absent in the testicular macrophages of young adult mice. Testicular macrophages did not display phagocytosis of the lipofuscin granules. In addition, the latter were not found in the intercellular spaces. These observations indicated that lipofuscin granules were formed, at least in a great part, within testicular macrophages as a consequence of metabolic changes occurring with age. Fine lamellar organization was seen in the lipofuscin granules of both Leydig cells and testicular macrophages. Frequently, lipofuscin granules originated from secondary lysosomes containing lipidic vacuoles only. Together with accumulation of the lipofuscin granules, changes of testicular macrophage fine morphology were observed. Endoplasmic reticulum and Golgi apparatus became poorly developed, and coated vesicles were rarely found. Fewer mitochondria were encountered, but their ultrastructure was not altered. These results suggest that in testicular macrophages lipofuscin accumulation is associated with a functional involution.     

Determination of amphetamine-derived designer drugs in human urine by SPE extraction and capillary electrophoresis with mass spectrometry detection

In recent years, a number of newer designer drugs have entered the illicit drug market. The methylenedioxy-derivates of amphetamine represent the largest group of designer drugs. This paper describes a method for screening for and quantification of ten 2,5-methylenedioxy-derivates of amphetamine and phenylethylamine in human urine, using capillary electrophoresis coupled to electrospray ionisation-mass spectrometry (CE-ESI-MS). Prior to CE-MS analysis, a simple solid-phase extraction (SPE) was used for sample cleanup. The method was validates according to international guidelines.     

Acrylamide-agarose copolymers: improved resolution of high molecular mass proteins in two-dimensional gel electrophoresis

A method was developed in order to analyse high molecular mass proteins by two-dimensional (2-D) electrophoresis using a copolymer of acrylamide and allyl agarose instead of Bis cross-linked polyacrylamide (PA) gels in sodium dodecyl sulphate-electrophoresis. In this work, the matrix composition was optimised to improve the resolution of proteins larger than 200 kDa. The new gel type does not entrap large proteins and protein complexes at the application site. Mechanical properties were investigated through rheological measurements, which suggested the formation of a highly entangled elastomeric soft gel. A high 2-D resolution of proteins, extracted from membranes of red blood cells, was obtained in these gels. An example of tryptic digestion, peptide extraction and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry was reported. The results demonstrate that the new gel is fully compatible with mass spectrometry protein analysis.     

Deep learning architectures for prediction of nucleosome positioning from sequences data

Background

Nucleosomes are DNA-histone complex, each wrapping about 150 pairs of double-stranded DNA. Their function is fundamental for one of the primary functions of Chromatin i.e. packing the DNA into the nucleus of the Eukaryote cells. Several biological studies have shown that the nucleosome positioning influences the regulation of cell type-specific gene activities. Moreover, computational studies have shown evidence of sequence specificity concerning the DNA fragment wrapped into nucleosomes, clearly underlined by the organization of particular DNA substrings. As the main consequence, the identification of nucleosomes on a genomic scale has been successfully performed by computational methods using a sequence features representation.

Results

In this work, we propose a deep learning model for nucleosome identification. Our model stacks convolutional layers and Long Short-term Memories to automatically extract features from short- and long-range dependencies in a sequence. Using this model we are able to avoid the feature extraction and selection steps while improving the classification performances.

Conclusions

Results computed on eleven data sets of five different organisms, from Yeast to Human, show the superiority of the proposed method with respect to the state of the art recently presented in the literature.

     

Annual flower strips support pollinators and potentially enhance red clover seed yield

Ecological intensification provides opportunity to increase agricultural productivity while minimizing negative environmental impacts, by supporting ecosystem services such as crop pollination and biological pest control. For this we need to develop targeted management solutions that provide critical resources to service‐providing organisms at the right time and place. We tested whether annual strips of early flowering phacelia Phacelia tanacetifolia support pollinators and natural enemies of seed weevils Protapion spp., by attracting and offering nectar and pollen before the crop flowers. This was expected to increase yield of red clover Trifolium pratense seed. We monitored insect pollinators, pests, natural enemies and seed yields in a total of 50 clover fields along a landscape heterogeneity gradient, over 2 years and across two regions in southern Sweden. About half of the fields were sown with flower strips of 125–2,000 m². The clover fields were pollinated by 60% bumble bees Bombus spp. and 40% honey bees Apis mellifera. The clover seed yield was negatively associated with weevil density, but was unrelated to bee species richness and density. Flower strips enhanced bumble bees species richness in the clover fields, with the strongest influence in heterogeneous landscapes. There were few detectable differences between crop fields with and without flower strips. However, long‐tongued bumble bees were redistributed toward field interiors and during phacelia bloom honey bees toward field edges. Clover seed yield also increased with increasing size of the flower strip. We conclude that annual flower strips of early flower resources can support bumble bee species richness and, if sufficiently large, possibly also increase crop yields. However, clover seed yield was mainly limited by weevil infestation, which was not influenced by the annual flower strips. A future goal should be to design targeted measures for pest control.