Chronic Inflammation Permanently Reshapes Tissue-Resident Immunity in Celiac Disease

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The Formation of Sugar Chains in Triterpenoid Saponins and Glycoalkaloids

Triterpenoid saponins and structurally related steroidal glycoalkaloids are a large and diverse family of plant glycosides. The importance of these compounds for chemical protection of plants against microbial pathogens and/or herbivores is now well-documented. Moreover, these compounds have a variety of commercial applications, e.g. as drugs or raw materials for pharmaceutical industry. Until recently there were only sparse data on the biosynthesis of saponins and glycoalkaloids, especially at the enzyme level. Substantial progress has recently been made, however, in our understanding of biosynthetic routes leading to the formation of the diverse array of aglycone skeletons found in these compounds as well as mechanisms of synthesis of their sugar moieties. This review highlights some of the advances made over past two decades in our understanding of the formation and modification of sugar moieties in triterpenoid saponins and glycoalkaloids.     

Laboratory trials to infect insects and nematodes by some acaropathogenic Hirsutella strains (Mycota: Clavicipitaceous anamorphs)

Laboratory assays have been carried out to artificially infect insect larvae of the birch bark-beetle (Scolytus ratzeburgi Jans.-Coleoptera, Scolytidae) and codling moth Cydia pomonella L. -Lepidoptera, Tortricidae) as well as the potato cyst nematode-Globodera rostochiensis Wollenweber, sugar beet nematode-Heterodera schachtii Schmidt and root-knot nematode-Meloidogyne hapla Chif (Nematoda, Heteroderidae), by the phialoconidia of some fungal species of the genus Hirsutella. From among four species tested on insects only H. nodulosa Petch infected about 20% of S. ratzeburgi larvae, whereas H. kirchneri (Rostrup) Minter, Brady et Hall, H. minnesotensis Chen, Liu et Chen, and H. rostrata Ba?azy et Wi?niewski did not affect insect larvae. Only single eggs of the root-knot nematode were infected by H. minnesotensis in the laboratory trials, whereas its larvae remained unaffected. No infection cases of the potato cyst nematode (G. rostochiensis) and sugar beet nematode eggs were obtained. Comparisons of DNA-ITS-region sequences of the investigated strains with GenBank data showed no differences between H. minnesotensis isolates from the nematodes Heterodera glycines Ichinohe and from tarsonemid mites (authors' isolate). A fragment of ITS 2 with the sequence characteristic only for H. minnesotensis was selected. Two cluster analyses indicated close similarity of this species to H. thompsonii as sister clades, but the latter appeared more heterogenous. Insect and mite pathogenic species H. nodulosa localizes close to specialized aphid pathogen H. aphidis, whereas the phytophagous mite pathogens H. kirchneri and H. gregis form a separate sister clade. Hirsutella rostrata does not show remarkable relations to the establishment of aforementioned groups. Interrelated considerations on the morphology, biology and DNA sequencing of investigated Hirsutella species state their identification more precisely and facilitate the establishment of systematic positions.     

Diversity of acaropathogenic fungi in Poland and other European countries

The occurrence, species diversity and some aspects of taxonomical affinity and host selectivity of acaropathogenic fungi associated with phytophagous, saprotrophic and predacious mites in Poland and other European countries were investigated on wild and cultivated plants, in insect feeding sites under the bark and in decayed wood. From among 33 species of fungi affecting mites only five species of Entomophthorales were separated and the most numerous were Neozygites floridana mostly on Tetranychus urticae, N. abacaridis on a few eriophyid species, and Conidiobolus coronatus attacking gamasid mites most frequently of the genus Dendrolaelaps. The most frequent mite pathogens occurring in mite communities on plants and in wood infested by insects were of the genus Hirsutella. Until now 13 of their form-species have been recognized in these habitats, but only H. kirchneri, H. necatrix and H. thompsonii (including its variety synnematosa) can be treated as exclusive oligophagous pathogens of phytophagous mites, though their potential host range seems to embrace only selected eriophyid or tarsonemid mites. Taxonomical differentiation of fungal strains was based on close morphological observations and molecular analysis of ITS region sequences. Two new species of acaropathogenic fungi were described in these studies. Hirsutella danubiensis sp. nov. was found in the tetranychid T. urticae, whereas H. vandergeesti sp. nov. affected phytoseiid mites of the genera Amblyseius, Neoseiulus, Seiulus and Typhlodromus, and the tarsonemid Tarsonemus lacustris.     

Antioxidative enzyme and glutathione S-transferase activities in diabetic rats exposed to long-term ASA treatment

Low-dose acetylsalicylic acid (ASA) treatment is a standard therapeutic approach in diabetes mellitus for prevention of long-term vascular complications. The aim of the present work was to investigate the effect of long-term ASA administration in experimental diabetes on activities of some liver enzymes: glutathione peroxidase (GSHPx), catalase, glucose-6-phosphate dehydrogenase (G6PDH) and glutathione S-transferase (GST). Blood glucose, glycated hemoglobin, as well as plasma ALT and AST activities increased in rats with streptozotocin-induced experimental diabetes. The long-term hyperglycemia resulted in decreased activities of GSHPx (by 26%), catalase (by 34%), GST (by 38%) and G6PDH (by 27%) in diabetic animals. We did not observe increased accumulation of membrane lipid peroxidation products or altered levels of reduced glutathione in livers. The linear correlation between blood glucose and glycated hemoglobin in diabetic animals was distorted upon ASA treatment, which was likely due to a chemical competition between nonenzymatic protein glycosylation and protein acetylation. The long-term ASA administration partially reversed the decrease in GSHPx activity, but did not influence the activities of catalase and GST in diabetic rats. Otherwise, some decrease in these parameters was noted in ASA-treated nondiabetic animals. Increased ASA-induced G6PDH activity was recorded in both diabetic and nondiabetic rats. While both glycation due to diabetic hyperglycemia and ASA-mediated acetylation had very similar effects on the activities of all studied enzymes but G6PDH, we conclude that non-enzymatic modification by either glucose or ASA may be a common mechanism of the observed convergence.     

Linear patterns of Alzheimer's disease mutations along alpha-helices of presenilins as a tool for PS-1 model construction

We performed an analysis of mutation patterns in all 10 hydrophobic regions (HRs) of presenilin-1 (PS-1) and PS-2 using a recent database of Alzheimer's disease (AD) mutations. The linear patterns were confirmed and extended to areas spanning as many as three faces of a given HR. The complementary areas of residues free of AD mutations were identified based on the location of non-pathogenic polymorphisms and PS-1 versus PS-2 amino acid discordances. Taking into account the location of areas of AD mutations and mutation-free areas/regions, we proposed a preliminary model of PS-1 structure using a general stick-out-mutation rule. To build a molecular structure of PS-1 and preserve features of the preliminary model, we used bacteriorhodopsin template in homology/comparative modelling. Two molecular models were built differing in the location of C-terminal fragment helices. The models properly distinguish residues belonging to AD-affected sites and non-pathogenic areas, and may be used for classification purposes. They also comply with experimental results, such as differences in accessibility of the catalytic residues in uncleaved PS-1, and binding of PEN-2 by the PS-1 NF motif.     

The construction of bifunctional fusion proteins consisting of MutS and GFP

MutS as a mismatch binding protein is a promising tool for SNP detection. Green fluorescent protein (GFP) is known as an excellent reporter domain. We constructed chimeric proteins consisting of MutS from Thermus thermophilus and GFPuv from Aequorea victoria by cloning the GFPuv gene into the plasmid vectors carrying the mutS gene. The GFPuv domain fused to the N-terminus of MutS (histag-GFP-MutS) exhibited the same level of green fluorescence as free GFPuv. To obtain the fluorescing histag-GFP-MutS protein the expression at 30 degrees C was required, while free GFPuv fluoresces when expressed both at 30 and 37 degrees C. The chimeric protein where the GFPuv domain was fused to the C-terminus of MutS exhibited much weaker green fluorescence (20-25% compared with those of histag-GFP-MutS or free GFPuv). The insertion of (ProGly)5 peptide linker between the MutS and GFP domains resulted in no significant improvement in GFP fluorescence. No shifts in the excitation and emission spectra have been observed for the GFP domain in the fusion proteins. The fusion proteins with GFP at the N- and C-terminus of MutS recognised DNA mismatches similarly like T. thermophilus MutS. The fluorescent proteins recognising DNA mismatches could be useful for SNP scanning or intracellular DNA analysis. The fusion proteins around 125 kDa were efficiently expressed in E. coli and purified in milligram amounts using metal chellate affinity chromatography.     

Decreased ER-associated degradation of alpha-TCR induced by Grp78 depletion with the SubAB cytotoxin

HeLa cells stably expressing the α chain of T-cell receptor (αTCR), a model substrate of ER-associated degradation (ERAD), were used to analyze the effects of BiP/Grp78 depletion by the SubAB cytotoxin. SubAB induced XBP1 splicing, followed by JNK phosphorylation, eIF2α phosphorylation, upregulation of ATF3/4 and partial ATF6 cleavage. Other markers of ER stress, including elements of ERAD pathway, as well as markers of cytoplasmic stress, were not induced. SubAB treatment decreased absolute levels of αTCR, which was caused by inhibition of protein synthesis. At the same time, the half-life of αTCR was extended almost fourfold from 70 min to 210 min, suggesting that BiP normally facilitates ERAD. Depletion of p97/VCP partially rescued SubAB-induced depletion of αTCR, confirming the role of VCP in ERAD of αTCR. It therefore appears that ERAD of αTCR is driven by at least two different ATP-ase systems located at two sides of the ER membrane, BiP located on the lumenal side, while p97/VCP on the cytoplasmic side. While SubAB altered cell morphology by inducing cytoplasm vacuolization and accumulation of lipid droplets, caspase activation was partial and subsided after prolonged incubation. Expression of CHOP/GADD153 occurred only after prolonged incubation and was not associated with apoptosis.     

Conformational studies of vasopressin and mesotocin using NMR spectroscopy and molecular modelling methods. Part II: Studies in the SDS micelle.

There is much evidence to support the hypothesis that lipids play a role in the interaction of peptide hormones with their membrane receptors. This interaction through change of peptide conformation can facilitate the entry of the hormone into the microenvironment of the receptor. In the present study we have examined the interaction of vasopressin and mesotocin with a lipid-sodium dodecylsulfate (SDS) micelle-using 2D nuclear magnetic resonance (NMR) and theoretical methods. Solution structures of two hormones in solution with SDS were established using the nuclear Overhauser effect (NOE) and the (3)J(NHHalpha) couplings. The amino acid sequences of these peptides are: c[C(1)-Y(2)-F(3)-Q(4)-N(5)-C(6)]-P(7)-R(8)-G(9)-NH(2) ([Arg(8)]vasopressin, AVP) and c[C(1)-Y(2)-I(3)-Q(4)-N(5)-C(6)]-P(7)-I(8)-G(9)-NH(2) (MT). Each of the peptides was found to occur as one stable conformation. AVP adopts the cis configuration on the Cys(1)-Tyr(2) peptide bond, a finding not reported so far. The three-dimensional structures of the two peptides studied were determined by a method that consisted of time-averaged molecular dynamics in an explicit SDS micelle with the parm99 force field in AMBER8.0 package. All calculated structures of the studied peptides form beta-turns in their cyclic parts. The C-terminal fragment of MT is bent, whereas that of AVP is extended.