Biomedical subjects

Galina Radeva

Publications and source records attributed to Galina Radeva.

6 recordsLinked to original sources

PubMed2006

Combined chemical and enzymatic stable isotope labeling for quantitative profiling of detergent-insoluble membrane proteins isolated using Triton X-100 and Brij-96.

Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five noncysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content.

Amino Acid Sequence↗

PubMed2005

Novel surface layer protein genes in Bacillus sphaericus associated with unusual insertion elements.

The surface layer (S-layer) protein genes of the uranium mining waste pile isolate Bacillus sphaericus JG-A12 and of its relative B. sphaericus NCTC 9602 were analysed. The almost identical N-termini of the two S-layer proteins possess a unique structure, comprising three N-terminal S-layer homologous (SLH) domains. The central parts of the proteins share a high homology and are related to the S-layer proteins of B. sphaericus CCM 2177 and P-1. In contrast, the C-terminal parts of the S-layer proteins of JG-A12 and NCTC 9602 differ significantly between each other. Surprisingly, the C-terminal part of the S-layer protein of JG-A12 shares a high identity with that of the S-layer protein of B. sphaericus CCM 2177. In both JG-A12 and NCTC 9602 the chromosomal S-layer protein genes are followed by a newly identified putative insertion element comprising three ORFs, which encode a putative transposase, a putative integrase/recombinase and a putative protein containing a DNA binding helix-turn-helix motif, and the S-layer-protein-like gene copies sllA (9602) or sllB (JG-A12). Interestingly, both B. sphaericus strains studied were found to contain an additional, plasmid-located and silent S-layer protein gene with the same sequence as sllA and sllB. The primary structures of the corresponding putative proteins are almost identical in both strains. The N-terminal and central parts of these S-layer proteins share a high identity with those of the chromosomally encoded functional S-layer proteins. Their C-terminal parts, however, differ significantly. These results strongly suggest that the S-layer protein genes have evolved via horizontal transfer of genetic information followed by DNA rearrangements mediated by mobile elements.

Bacillus↗

PubMed2005

P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains.

P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily responsible for the ATP-driven extrusion of diverse hydrophobic molecules from cells, is a cause of multidrug resistance in human tumours. Pgp can also operate as a phospholipid and glycosphingolipid flippase, and has been functionally linked to cholesterol, suggesting that it might be associated with sphingolipid-cholesterol microdomains in cell membranes. We have used nonionic detergent extraction and density gradient centrifugation of extracts from the multidrug-resistant Chinese hamster ovary cell line, CH(R)B30, to address this question. Our data indicate that Pgp is localized in intermediate-density membrane microdomains different from classical lipid rafts enriched in Src-family kinases. We demonstrate that Brij-96 can selectively isolate the Pgp domains, separating them from the caveolar and classical lipid rafts. Pgp was found entirely in the Brij-96-insoluble domains, and only partially in the Triton X-100-insoluble membrane microdomains. We studied the sensitivity of these domains to cholesterol removal, as well as their relationship to GM(1) ganglioside- and caveolin-1-enriched caveolar domains. We found that the buoyant density of the Brij-96-based Pgp-containing microdomains was sensitive to cholesterol removal by methyl-beta-cyclodextrin. The Brij-96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X-100-based caveolin-1/GM(1) microdomains did not. Using confocal fluorescence microscopy, we determined that caveolin-1 and GM(1) colocalized, while Pgp and caveolin-1, or Pgp and GM(1), did not. Our results suggest that Pgp does not interact directly with caveolin-1, and is localized in intermediate-density domains, distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Bacterial diversity in water samples from uranium wastes as demonstrated by 16S rDNA and ribosomal intergenic spacer amplification retrievals.

Bacterial diversity was assessed in water samples collected from several uranium mining wastes in Ger many and in the United States by using 16S rDNA and ribosomal intergenic spacer amplification retrievals. The results obtained using the 16S rDNA retrieval showed that the samples collected from the uranium mill tailings of Schlema/Alberoda, Germany, were predominated by Nitrospina-like bacteria, whereas those from the mill tailings of Shiprock, New Mexico, USA, were predominated by gamma-Pseudomonas and Frauteria spp. Additional smaller populations of the Cytophaga-Flavobacterium-Bacteroides group and alpha- and delta-Proteobacteria were identified in the Shiprock samples as well. Proteobacteria and Cytophaga-Flavobacterium-Bacteroides were also found in the third uranium mill tailings studied, Gittersee/Coschütz, Germany, but the groups of the predominant clones were rather small. Most of the clones of the Gittersee/Coschütz samples represented individual sequences, which indicates a high level of bacterial diversity. The samples from the fourth uranium waste studied, Steinsee Deponie B1, Germany, were predominantly occupied by Acinetobacter spp. The ribosomal intergenic spacer amplification retrieval provided results complementary to those obtained by the 16S rDNA analyses. For instance, in the Shiprock samples, an additional predominant bacterial group was identified and affiliated with Nitrosomonas sp., whereas in the Gittersee/Coschütz samples, anammox populations were identified that were not retrieved by the applied 16S rDNA approach.

Bacteria↗

PubMed2004

Isolation and characterization of lipid rafts with different properties from RBL-2H3 (rat basophilic leukaemia) cells.

Lipid rafts are plasma-membrane microdomains that are enriched in certain lipids (sphingolipids, glycosphingolipids and cholesterol), as well as in lipid-modified proteins. Rafts appear to exist in the liquid-ordered phase, which contributes to their partitioning from the surrounding liquid-disordered glycerophospholipid environment. DRM (detergent-resistant membrane) fractions isolated from cells are believed to represent coalesced lipid rafts. We have employed extraction using two different non-ionic detergents, Brij-96 and Triton X-100, to isolate detergent-resistant lipid rafts from rat basophilic leukaemia cell line RBL-2H3, and compared their properties with each other and with plasma-membrane vesicles. DRM fractions were isolated as sealed unilamellar vesicles of similar size (135-170 nm diameter), using either sucrose-density-gradient sedimentation or gel-filtration chromatography. Lipid rafts isolated using Brij-96 and Triton X-100 differed in density, protein content and the distribution between high- and low-density fractions of the known raft constituents, Thy-1, and the non-receptor protein tyrosine kinases, Yes and Lyn. Lyn was found in the raft microdomains in predominantly phosphorylated form. The level of enrichment of the protein constituents of the isolated lipid rafts seemed to depend on the ratio of cell lipid/protein to detergent. As indicated by reactivity with anti-Thy-1 antibodies, lipid rafts prepared using Brij-96 appeared to consist of vesicles with primarily right-side-out orientation. Both Brij-96 and Triton X-100 appear to isolate detergent-insoluble raft microdomains from the rat basophilic leukaemia cell line RBL-2H3, but the observed differences suggest that either the detergents themselves play a role in determining the physicochemical characteristics of the resulting DRM fractions, or different subsets of rafts are isolated by the two detergents.

Animals↗

PubMed2004

GPI-anchored protein cleavage in the regulation of transmembrane signals.

The structure of covalently-linked glycosylphosphatidylinositol (GPI) anchors of membrane proteins displayed on the cell surface is described. Evidence of how the GPI-anchors are sorted into membrane rafts in the plasma membrane is reviewed. Proteins are released by hydrolysis of the linkage to the GPI anchor and phospholipases from different sources involved in this process are characterised. The regulation of protein conformation and function resulting from phospholipase cleavage of the GPI anchor is discussed in the context of its role in signal transduction by insulin. In this signalling system, re-distribution of critical membrane components, including GPI-anchored proteins and non-receptor tyrosine kinases, between different raft domains appears to play a central role in the signal transduction pathway.

Animals↗