Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process

Tissue transglutaminase (TG2) can modify proteins by transamidation or deamidation of specific glutamine residues. TG2 has a major role in the pathogenesis of celiac disease as it is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides that are recognized by CD4(+), DQ2-restricted T cells from the celiac lesions. Capillary electrophoresis with fluorescence-labeled gliadin peptides was used to separate and quantify deamidated and transamidated products. In a competition assay, the affinity of TG2 to a set of overlapping gamma-gliadin peptides was measured and compared with their recognition by celiac lesion T cells. Peptides differed considerably in their competition efficiency. Those peptides recognized by intestinal T cell lines showed marked competition indicating them as excellent substrates for TG2. The enzyme fine specificity of TG2 was characterized by synthetic peptide libraries and mass spectrometry. Residues in positions -1, +1, +2, and +3 relative to the targeted glutamine residue influenced the enzyme activity, and proline in position +2 had a particularly positive effect. The characterized sequence specificity of TG2 explained the variation between peptides as TG2 substrates indicating that the enzyme is involved in the selection of gluten T cell epitopes. The enzyme is mainly localized extracellularly in the small intestine where primary amines as substrates for the competing transamidation reaction are present. The deamidation could possibly take place in this compartment as an excess of primary amines did not completely inhibit deamidation of gluten peptides at pH 7.3. However, lowering of the pH decreased the reaction rate of the TG2-catalyzed transamidation, whereas the rate of the deamidation reaction was considerably increased. This suggests that the deamidation of gluten peptides by TG2 more likely takes place in slightly acidic environments.     

Enhanced values of the RBE and H ratio for cytogenetic effects induced by secondary electrons from an X-irradiated gold gurface

The low-energy secondary electrons emerging from the entrance surface of an X-irradiated gold foil increase the dose to cells in contact with or at micrometer distances from this surface (Radiat. Res. 150, 92-100, 1998). We examined the effect of the spectrum of these low-energy electrons on the RBE for cytogenetic effects and showed that this RBE was increased. A monolayer of surface-attached human T lymphocytes was exposed to 60 kV X rays in the absence or presence of a gold foil positioned immediately behind the cell layer or separated from it by a Mylar foil 0.9 or 2 microm thick. The enhancement of dose in the cell nuclei caused by the photoelectrons and Auger electrons emerging from the entrance surface of the gold foil was measured by TSEE dosimetry. Dose enhancement factors of 55.7, 46.6 and 37.5 were obtained with 0, 0.9 and 2 microm of Mylar inserted between the gold surface and the cell layer. This large enhancement results from the photoelectric effect in the gold foil, as shown by the accompanying Monte Carlo calculations of the secondary electron spectra at the gold surface. Auger electrons from the gold foil generally were not able to penetrate into the cell nuclei except for that fraction of the cells that had a very thin (< 0.7 microm) layer of cytoplasm and membranes between gold surface and cell nucleus. The dose-yield curves for dicentric chromosomes plus centric rings and for acentric fragments obtained after exposures without or with the gold foil were linear-quadratic. The coefficient alpha, the slope of the linear yield component, was increased in the presence of the gold foil and showed RBE values ranging from 1.7 to 2.2 compared to exposures in absence of the gold foil. The ratio of the yield of interstitial deletions and dicentrics (H ratio) was significantly increased from about 0.17 in the absence of the gold foil to about 0.22 in the presence of the gold foil. The increases in the RBE and the H ratio are interpreted in microdosimetric terms: The preferred occurrence of electron track ends in the vicinity of the gold surface causes an increase in the dose-mean restricted linear energy transfer in cell nuclei exposed to the photoelectrons and Auger electrons.     

Novel locus required for expression of high-level macrolide-lincosamide-streptogramin B resistance in Staphylococcus aureus

The yycF1(Ts) mutation in Staphylococcus aureus conferred hypersensitivity to macrolide-lincosamide-streptogramin B (MLS(B)) antibiotics on strains either containing or lacking ermB. The overexpression of the S. aureus Ssa protein restored the yycF1 mutant to wild-type levels of susceptibility. Inactivation of ssa in an unmutagenized strain dramatically reduced ermB-based resistance. Conditional loss of function or expression of ssa in the yycF1 mutant is proposed to result in the observed hypersensitivity to MLS(B) antibiotics.     

Stimulation of oxygen evolution in photosystem II by copper(II) ions

We have found that copper(II) ions at about equimolar Cu2+/photosystem II (PS II) reaction center proportions stimulate oxygen evolution nearly twofold. This high affinity Cu-binding site is different from the binding sites of Mn and Ca ions. The analysis of the Cu2+ content in PS II preparations isolated from wild-type tobacco and a tobacco mutant deficient in light-harvesting complex suggests that Cu2+ may be a native component of PS II and may take part in the oxygen evolution process. At higher concentrations, Cu2+ ions inhibit oxygen evolution and quench fluorescence.     

Influence of different light intensities on the content of diosgenin, lipids, carotenoids and fatty acids in leaves of Dioscorea zingiberensis

Cultivation of the climbing plant Dioscorea zingiberensis at a light intensity of 100 microE. m(-2) sec(-1) yields three different phenotypes. Most of the plants grow as green phenotype (DzW). Two further forms differ in their leaf shape and leaf color. Whereas one type exhibits a more pointed leaf shape in the upper part of the plant with leaves appearing yellow-green with white stripes or hatchings (DzY), the other type shows a more round leaf shape with an intensive yellow-green color (DzT). These three plant types differ in their diosgenin content not only in their rhizomes but also in the chloroplasts. In the rhizomes the diosgenin content in the green form is 0.4%, in the DzY-form 0.6% and in the DzT-form even 1.3% of the dry weight. Furthermore, even in chloroplasts of the green DzW-form and of the DzY-form the presence of diosgenin was demonstrated. It occurs there as the epimeric form yamogenin. The DzT-form contains no yamogenin in its chloroplasts. Besides this, these plant forms differ in their chlorophyll and carotenoid content and in their fatty acid composition. Carotenoids increase from 1.3% of total lipids in the green phenotype to 3.3% in the DzY- and to 4.2% in the DzT-form. This increase refers to beta-carotene as well as to lutein and neoxanthin. The chlorophyll content in the green type is 8.1% and lower in the DzY-form with 7%. The highest chlorophyll content is found in the DzT-form with 12%. Fatty acids in the DzY-form and in the DzT-form have a more unsaturated character than in the green phenotype. The content of the monoenoic acid trans-hexadecenoic acid is considerably lower in both phenotypes when compared to the green phenotype. In both phenotypes the quantity of fatty acids with 16 carbon atoms is reduced, whereas fatty acids with 18 carbon atoms occur in higher concentration. Cultivation of the green phenotype (DzW) at the three light intensities of 10, 100 and 270 microE x m(-2) x sec(-1) leads to changes of the diosgenin content in rhizomes, to an increase of leaf dry weight, to a reduction of the grana structure in chloroplasts and therewith to a decrease of the chlorophyll content. The total lipid content is highest under the cultivation at 100 microE x m(-2) x sec(-1) and reduced by 30% at 10 and 270 microE x m(-2) x sec(-1). Carotenoids, however, are highest in shaded plants (10 microE x m(-2) x sec(-1)) and plants grown under high light conditions of 270 microE x m(-2) x sec(-1). At 100 microE x m(-2) x sec(-1) a decrease of saturated fatty acids is observed in comparison to plants grown under shaded conditions.     

Biotransformations using prokaryotic P450 monooxygenases

Recent studies on microbial cytochrome P450 enzymes have covered several new areas. Advances have been made in structure-function analysis and new non-enzymatic/electrochemical systems for the replacement of NAD(P)H in biocatalysis have been developed. Furthermore, the properties of some enzymes have been re-engineered by site-directed mutagenesis or by methods of directed evolution and new P450s have been functionally expressed and characterized. It is thought that a combination of these approaches will facilitate the use of isolated P450 monooxygenases in biocatalysis.     

Effects of higher-order nuclear structure and Rad51 overexpression on radiation-induced chromosome rearrangements

The microchromosomes (MICs) in chicken DT40 lymphocytes are usually clustered in the center of the nucleus, whereas the macrochromosomes (MACs) are preferentially located toward the nuclear periphery. This compartmentalized architecture of the nucleus is associated with a low frequency of translocations between MICs and MACs after induction of DNA breaks by a radiation track(s). In contrast, the MICs in chick embryo fibroblasts (CEFs) tend to be located throughout the entire nuclear volume. The resulting side-to-side arrangement of MIC and MAC territories favors radiation-induced MIC/MAC translocations, which occur more frequently in CEF cells than MIC/MIC or MAC/MAC rearrangements. Collectively, our results suggest that preformed physical contacts are a prerequisite for the generation of chromosome rearrangements through recombinational repair of DNA damage. Cell type-specific higher-order nuclear organization may prevent or stimulate the formation of particular chromosome aberrations in pathology and evolution. Ectopic expression of the recombination protein Rad51 can protect cells from radiation-induced translocations. The repair activity of overexpressed Rad51 is more important for cells that are irradiated in S/G(2) phase than for cells in G(1) phase. Evidently, homologous recombination between sister chromatids of a replicated chromosome is more frequent than that between homologous or heterologous chromosomes during G(1) phase.     

Comparative mapping of Z-orthologous genes in vertebrates: implications for the evolution of avian sex chromosomes

Sex chromosomes of birds and mammals are highly differentiated and share several cytological features. However, comparative gene mapping reveals extensive conserved synteny between the chicken Z sex chromosome and human chromosome 9 but not the human X sex chromosome, implying an independent origin of avian and mammalian sex chromosomes. To better understand the evolution of the avian Z chromosome we analysed the synteny of chicken Z-linked genes in zebrafish, which is the best-mapped teleost genome so far. Existing zebrafish maps do not support the existence of an ancestral Z linkage group in the zebrafish genome, whereas mammalian X-linked genes show at least some degree of synteny conservation. This is consistent with in situ hybridisation mapping data in the freshwater pufferfish, Tetraodon nigroviridis where mammalian X-linked genes show a much higher degree of conserved synteny than human chromosome 9 or the avian Z chromosome. Collectively, these data argue in favour of a more recent evolution of the avian Z chromosome, compared with the mammalian X.     

Days and knights discussing membrane dynamics in endocytosis: meeting report from the Euresco/EMBL Membrane Dynamics in Endocytosis, 6--11 October in Tomar, Portugal

The Euresco/EMBL sponsored meeting on 'Membrane Dynamics in Endocytosis' took place on 6–11 October in Tomar, Portugal. Here we report on the 5 full days of exciting talks and active poster sessions that covered topics ranging from the mechanisms of clathrin-mediated endocytosis, the regulation of phagocytosis, caveolae dynamics and function, the role of lipids in regulating endocytic transport, the formation of and sorting into and out of multivesicular bodies, new links between the actin cytoskeleton and vesicular transport, and emerging roles for endocytic trafficking in signal transduction and development.