Assessment of N-glycan heterogeneity of cactus glycoproteins by one-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Artificial environmental conditions in tissue culture, such as elevated relative humidity and rich nutrient medium, can influence and modify tissue growth and induce spontaneous changes from characteristic organization pattern to unorganized callus. As succulent plants with crassulacean acid metabolism, cacti are particularly susceptible to this altered growth environment. Glycosylated proteins of Mammillaria gracillis tissues cultivated in vitro, separated by SDS-PAGE, were detected with Con A after the transfer of proteins onto the nitrocellulose membrane. The glycan components were further characterized by affinity blotting with different lectins (GNA, DSA, PNA, and RCA(120)). The results revealed significant differences in glycoprotein pattern among the investigated cactus tissues (shoot, callus, hyperhydric regenerant, and tumor). To test whether the N-glycosylation of the same protein can vary in different developmental stages of cactus tissue, the N-glycans were analyzed by MALDI-TOF MS after in-gel deglycosylation of the excised 38-kDa protein band. Paucimannosidic-type N-glycans were detected in oligosaccharide mixtures from shoot and callus, while the hyperhydric regenerant and tumor shared glycans of complex type. The hybrid oligosaccharide structures were found only in tumor tissue. These results indicate that the adaptation of plant cells to artificial environment in tissue culture is reflected in N-glycosylation, and structures of N-linked glycans vary with different developmental stages of Mammillaria gracillis tissues.     

Comparison of verdeluz orange G and modified Gallego stains

Tumors of the oral cavity include combinations of hard and soft tissues that may be difficult to identify using routine hematoxylin and eosin (H & E) staining. Although combination stains can demonstrate hard and soft tissues, trichrome stains, such as VanGieson and Masson, cannot differentiate dental hard tissues, such as dentin, cementum and osteoid. Modified Gallegos (MGS) and verdeluz orange G-acid fuchsin (VOF) stains can differentiate components of teeth. We used 10 tissue sections of decalcified bone and 10 pathologic tissue sections that contained different calcified tissues including peripheral ossifying fibroma, odontoma, central ossifying fibroma and cemento-ossifying fibroma. Sections were stained with H & E, VOF or MGS. H and E stained both hard tissues pink. VOF stained bone purple-red, cementum red and collagen blue. MGS stained bone green-blue, cementum red and collagen blue. VOF staining intensity and differentiation was better than MGS staining. VOF staining demonstrated hard tissue components distinctly and exhibited good contrast with the surrounding connective tissue. VOF also is a simple, single step, rapid staining procedure.     

Legionella pneumophila in Thermal Spa – Methodological Aspects of Sampling and Analysis

The aim of the study was to check the occurrence of Legionella pneumophila in thermal water and to compare different analytical methods of its determination and/or quantification used in different laboratories. The research has shown that there are noticeable differences between results from culture method, qPCR, and FISH. Significant influence on the results has got analysts qualification, samples preparation, and equipment parameters. Very important is also the homogeneousness of the samples. qPCR or FISH method can be used as a preliminary, quick study, for identification of Legionella in water samples but the results obtained should be confirmed by culture method.     

Levansucrases from Pseudomonas syringae pv. tomato and P. chlororaphis subsp. aurantiaca: substrate specificity, polymerizing properties and usage of different acceptors for fructosylation

Levansucrases of Pseudomonas syringae pv. tomato DC3000 (Lsc3) and Pseudomonas chlororaphis subsp. aurantiaca (also Pseudomonas aurantiaca) (LscA) have 73% identity of protein sequences, similar substrate specificity and kinetic properties. Both enzymes produce levan and fructooligosaccharides (FOS) of varied length from sucrose, raffinose and sugar beet molasses. A novel high-throughput chip-based nanoelectrospray mass spectrometric method was applied to screen alternative fructosyl acceptors for levansucrases. Lsc3 and LscA could both transfructosylate d-xylose, d-fucose, l- and d-arabinose, d-ribose, d-sorbitol, xylitol, xylobiose, d-mannitol, d-galacturonic acid and methyl-α-d-glucopyranoside and heterooligofructans with degree of polymerization up to 5 were detected. The ability of d-sorbitol, xylobiose, d-galacturonic acid, d-mannitol, xylitol and methyl-α-d-glucopyranoside to serve as fructosyl acceptors for levansucrases is shown for the first time. Expectedly, site-directed mutagenesis of His321 in Lsc3 to Arg, Lys, Leu and Ser resulted in proteins with decreased catalytic activity, affinity for sucrose and polymerizing ability. Random mutagenesis yielded a Lsc3 mutant Thr302Pro with reduced synthesis of levan and long-chain FOS. Thr302 is located in conserved DQTERP region of levansucrases adjacent to predicted acid-base catalyst Glu303. Thr302 and His321 are predicted to belong to +1 subsite of the substrate binding region of Lsc3.     

Mass spectrometry of gangliosides in extracranial tumors: Application to adrenal neuroblastoma

We report here on the introduction of mass spectrometry (MS) for profiling of native gangliosides from an extracranial tumor. The analytical approach was based on a modern platform combining the superior sensitivity and reproducibility of fully automated chip-based nanoelectrospray ionization (nanoESI) with the high resolution and mass accuracy provided by a hybrid quadrupole time-of-flight (QTOF) instrument. The feasibility of the method for the analysis of gangliosides, which are much less expressed in extracranial tissues, was here tested using as the model substrate an adrenal neuroblastoma (NB) specimen located in the abdominal region of a 2-year-old infant. Under properly optimized conditions, MS profiling revealed information on at least 61 different gangliosides exhibiting heterogeneity of the glycan and lipid compositions. NB was found dominated by species bearing short-chain oligosaccharide cores with a reduced overall Neu5Ac content. By chip–nanoESI MS, preceding findings related to the GD2 role in NB were confirmed. Moreover, the screening experiments offered novel information supporting the possible biomarker role of GM4, GM3, and GM1 ganglioside classes. Structural analysis of GM1(d18:1/18:2) and GD1(d18:0/19:0) possibly tumor-associated markers, carried out by tandem MS (MS/MS) using collision-induced dissociation (CID) at low energies, indicated that both GM1a and GD1b isomers are present in NB.     

Analysis of novel over‐ and under‐sulfated glycosaminoglycan sequences by enzyme cleavage and multiple stage MS

We report on a novel strategy for identification of specific sulfation motifs in chondroitin/dermatan sulfate (CS/DS) chain derived from decorin (Dcn), based on enzyme cleavage and multistage MS (MSⁿ). Released CS/DS chains were digested with chondroitin B and in parallel with AC I lyases to obtain oligosaccharides of known hexuronic acid (HexA) epimerization. The depolymerized chains were separated by gel filtration, and collected di‐ and hexasaccharides were analyzed by ESI MSⁿ. MS² on bisulfated 4,5‐Δ‐HexAGalNAc revealed an additional sulfate ester group at 4,5‐Δ‐HexA. MS² data provided evidence upon GlcA sulfation in Dcn due to the fact that 4,5‐Δ‐HexA derived from GlcA after chondroitin AC I lyase treatment. Hexasaccharide screening in the MS¹ mode indicated direct correlation between the sulfate distribution and HexA epimerization. MSⁿ performed on ions that, according to mass calculation, correspond to pentasulfated [4,5‐Δ‐HexAGalNAc(GlcAGalNAc)₂], trisulfated [4,5‐Δ‐HexAGalNAc(GlcAGalNAc)₂] with IdoA‐derived 4,5‐Δ‐HexA at the nonreducing end, tetrasulfated [4,5‐Δ‐HexAGalNAc(IdoAGalNAc)₂] and monosulfated [4,5‐Δ‐HexAGalNAc(IdoAGalNAc)₂] with GlcA‐derived 4,5‐Δ‐HexA at the nonreducing end rendered fragmentation patterns confirming the presence of over‐, regular, and under‐sulfated regions as well as structural motifs having both types of HexA sulfated within Dcn CS/DS.     

Defining the antigen receptor-dependent regulatory network that induces arrest of cycling immature B-lymphocytes

Background  

Engagement of the antigen receptor on immature B-lymphocytes leads to cell cycle arrest, and subsequent apoptosis. This is an essential process for eliminating self reactive B cells during its different stages of development. However, the mechanism by which it is achieved is not completely understood.     

Effects of bryophytes and lichens on seedling emergence of alvar plants: evidence from greenhouse experiments

Emergence of seedlings of four alvar grassland species ( Arenaria serpyllifolia , Festuca ovina , Filipendula vulgaris and Veronica spicata ) in bryophyte and lichen carpets was analysed in a series of greenhouse experiments. The aspects investigated were: the influence of thickness of moss mats, both in dry and moist conditions, the effects of thick Cladonia spp. clumps, and of living vs dead moss shoots and lichen podetia. Overall, Festuca seedlings emerged best whereas the small-seeded species, Arenaria and Veronica , had the lowest emergence. Moisture had a significant effect only on the emergence of Festuca seedlings, which emerged better in the dry treatment than in the moist. A thick moss cover negatively affected seedling emergence of Arenaria and Veronica but did not affect the emergence of Festuca . Filipendula showed lower seedling emergence in both thick and thin moss than on bare soil only in the dry treatment, whereas in the moist treatment emergence did not differ among the three substrates. Arenaria seedlings emerged less in thick and thin moss than on bare soil in the dry treatment, whereas in the moist treatment emergence in the thin moss was not different from bare soil. Thus, in relatively dry environments even a thin moss cover may inhibit rather than facilitate seedling emergence. The lichen clumps inhibited only the emergence of the forbs. Both living moss shoots and lichen podetia inhibited emergence of Veronica seedlings but did not affect Festuca . In contrast, emergence in the presence of dead mosses and lichens did not differ from emergence in their absence for both species. Hence, inhibition of seedling emergence by bryophytes and lichens of at least some vascular plant species may be mediated by some biotic factor. However, the effect of differences in substrate properties on germination cannot be excluded     

SUPFAM: A database of sequence superfamilies of protein domains

Background  

SUPFAM database is a compilation of superfamily relationships between protein domain families of either known or unknown 3-D structure. In SUPFAM, sequence families from Pfam and structural families from SCOP are associated, using profile matching, to result in sequence superfamilies of known structure. Subsequently all-against-all family profile matches are made to deduce a list of new potential superfamilies of yet unknown structure.     

Immunohistochemical localization of TLR2 and CD14 in gingival tissue of healthy individuals and patients with chronic periodontitis

We used immunohistochemistry to quantify and compare the expression of Toll-like receptor 2 (TLR2) and cluster of differentiation 14 (CD14) in gingival tissues of both healthy individuals and patients with chronic periodontitis. We also correlated the expression of TLR2 and CD14 with the histological grades of chronic periodontitis. We examined 30 gingival specimens from chronic periodontitis patients and 10 from healthy individuals. Tissues from both groups were immunostained with antibodies against TLR2 and CD14. TLR2 and CD14 were expressed by endothelial cells, fibroblasts, lymphocytes and plasma cells. The immunohistochemical expression of TLR2 and CD14 was significantly greater in inflammatory cells of the chronic periodontitis group than in healthy individuals. Expression of these molecules was greater in the inflammatory cells of connective tissue adjacent to pocket epithelium in both groups. The expression of TLR2 and CD14 was greatest in the periodontitis group that was classified as severe grade, followed by moderate and mild grades, which suggests a role of TLR2 and CD14 in the pathogenesis of chronic periodontitis. The positive correlation of TLR2 and CD14 expression levels with the severity grades of chronic periodontitis suggests that they are correlated also with disease severity; therefore, they may be useful for predicting disease progression. Our findings are consistent with the possibility that CD14 acts as a co-receptor for TLR2.