Eco-morphological studies on pleopodal lungs and cuticle in Armadillidium species (Crustacea,Isopoda, Oniscidea)

Terrestrial isopods (Crustacea, Isopoda, Oniscidea) have adapted to land life by diverse morphological, physiological and behavioral changes. Woodlice species exhibit a large variety in this respect, their preferences ranging from moist to dry habitats. These moisture preference values are related to various morphological adaptations, rendering terrestrial isopods amenable to studying morphological adaptations to terrestrial life. We performed a comparison of four Armadillidium species (Armadillidium zenckeri, Armadillidium nasatum, Armadillidium versicolor, Armadillidium vulgare), by quantifying two morphological traits: the extent of the interfacial endothelium between the respiratory space and the hemolymph within pleopodal lungs and the thickness of tergite cuticle, which are ‘key factors’ in determining protection from desiccation. These values were measured from light micrographs of cross-sectioned lungs. The cosmopolitan A. vulgare, as a habitat generalist, seems to be the most resistant against desiccation and other environmental conditions, while A. zenckeri is the most sensitive one. Light microscopic studies revealed that the four species can be ordered similarly, if we compare them by the extension of the endothelial interface and cuticle thickness, suggesting that these morphological traits are important determinants of their distribution on habitat, microhabitat scales and through the existence of suitable habitats – together with many other factors – the geographical pattern of species occurence.     

Silicon: The key element in early stages of biocalcification

Biocalcification is a widespread process of forming hard tissues like bone and teeth in vertebrates. It is also a topic connecting life sciences and earth sciences: calcified skeletons and shells deposited as sediments represent the earth's fossil record and are of paramount interest for biogeochemists trying to get an insight into the past of our planet. This study reports on the role of silicon in the early biocalcification steps, where silicon and calcium were detected on the surface of cyanobacteria (initial stage of lacustrine calcite precipitation) and in crustacean cuticles. By using innovative methodological approaches of correlative microscopy (AFM in combination with analytical TEM: EFTEM, EELS) the chemical form of silicon in biocalcifying matrices and organic-inorganic particles is determined. Previously, silicon was reported to be localized in active growth areas in the young bone of vertebrates. We have found evidence that biocalcification in evolutionarily distant organisms involves very similar initial phases with silicon as a key element at the organic-inorganic interface.     

Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides

Protein glycosylation was explored by direct nanoESI MS and MS/MS analysis of ZIC-HILIC-enriched proteolytic glycopeptides without further separation or purification. In a previous publication, we demonstrated that a direct MS-based analysis of proteolytic glycopeptides is feasible for a number of proteins (Henning , S. J. Mass Spectrom. 2007 , 42 , 1415 - 21). This method has now been refined for two aspects: (1) separation of glycopeptides by use of ZIC-HILIC SPE and (2) the use of unspecific proteases like thermolysin, elastase, or a trypsin/chymotrypsin mixture leading per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. Furthermore, the glycopeptides produced by the above proteases in general contain short peptide backbones thus improving-probably due to their higher hydrophilicity--the ZIC-HILIC-based separation. The combination of unspecific proteolysis, glycopeptide separation, and their direct MS analysis was successfully accomplished for probing glycoproteins carrying high-mannose type (ribonuclease B), neutral (asialofetuin), and acidic (haptoglobin and α1-acid glycoprotein) complex type glycans as well as for glycopeptides derived from glycoprotein mixtures and, finally, for exploring the glycosylation of a human IgG preparation. Our results show that the presented method is a fast, facile, and inexpensive procedure for the elucidation of protein N-glycosylation.     

Cytotoxic activity of sesquiterpene lactones from Inula britannica on human cancer cell lines

Five new sesquiterpene lactones (1–5) were isolated from Inula britannica collected in the wild from Serbia along with five known compounds (6–10). Sesquiterpene lactones were isolated using centrifugal partition chromatography followed by combination of flash chromatography and semi-preparative HPLC. Isolated compounds were screened for cytotoxic activity on four different human cancer cell lines and their multi-drug resistant counterparts, as well as on normal human keratinocytes. Sesquiterpene lactones showed similar cytotoxic activity toward drug sensitive and drug resistant cancer cell lines.     

Evidence for Distinct Functions of MRE11 in Arabidopsis Meiosis

The evolutionary conserved Mre11/Rad50/Nbs1 complex functions as one of the guardians of genome integrity in eukaryotes; it is required for the double-strand break repair, meiosis, DNA checkpoint, and telomere maintenance. To better understand the role of the MRE11 gene in Arabidopsis, we performed comparative analysis of several mre11 alleles with respect to genome stability and meiosis. The mre11-4 and mre11-2 alleles presumably produce truncated MRE11 proteins composed of the first 499 and 529 amino acids, respectively. Although the putative MRE11 truncated proteins differ only by 30 amino acids, the mutants exhibited strikingly different phenotypes in regards to growth morphology, genome stability and meiosis. While the mre11-2 mutants are fully fertile and undergo normal meiosis, the mre11-4 plants are sterile due to aberrant repair of meiotic DNA breaks. Structural homology analysis suggests that the T-DNA insertion in the mre11-4 allele probably disrupted the putative RAD50 interaction and/or homodimerization domain, which is assumed to be preserved in mre11-2 allele. Intriguingly, introgression of the atm-2 mutant plant into the mre11-2 background renders the double mutant infertile, a phenotype not observed in either parent line. This data indicate that MRE11 partially compensates for ATM deficiency in meiosis of Arabidopsis.     

Characterization of Lactobacillus brevis L62 strain, highly tolerant to copper ions

Lactic acid bacteria (LAB) as starter culture in food industry must be suitable for large-scale industrial production and possess the ability to survive in unfavorable processes and storage conditions. Approaches taken to address these problems include the selection of stress-resistant strains. In food industry, LAB are often exposed to metal ions induced stress. The interactions between LAB and metal ions are very poorly investigated. Because of that, the influence of non-toxic, toxic and antioxidant metal ions (Zn, Cu, and Mn) on growth, acid production, metal ions binding capacity of wild and adapted species of Leuconostoc mesenteroides L3, Lactobacillus brevis L62 and Lactobacillus plantarum L73 were investigated. The proteomic approach was applied to clarify how the LAB cells, especially the adapted ones, protect themselves and tolerate high concentrations of toxic metal ions. Results have shown that Zn and Mn addition into MRS medium in the investigated concentrations did not have effect on the bacterial growth and acid production, while copper ions were highly toxic, especially in static conditions. Leuc. mesenteroides L3 was the most efficient in Zn binding processes among the chosen LAB species, while L. plantarum L73 accumulated the highest concentration of Mn. L. brevis L62 was the most copper resistant species. Adaptation had a positive effect on growth and acid production of all species in the presence of copper. However, the adapted species incorporated less metal ions than the wild species. The exception was adapted L. brevis L62 that accumulated high concentration of copper ions in static conditions. The obtained results showed that L. brevis L62 is highly tolerant to copper ions, which allows its use as starter culture in fermentative processes in media with high concentration of copper ions.     

Macrolactonolides: A novel class of anti-inflammatory compounds

A new concept in design of safe glucocorticoid therapy was introduced by conjugating potent glucocorticoid steroids with macrolides (macrolactonolides). These compounds were synthesized from various steroid 17β-carboxylic acids and 9a-N-(3-aminoalkyl) derivatives of 9-deokso-9a-aza-9a-homoeritromicin A and 3-descladinosyl-9-deokso-9a-aza-9a-homoeritromicin A using stable alkyl chain. Combining property of macrolides to preferentially accumulate in immune cells, especially in phagocyte cells, with anti-inflammatory activity of classic steroids, we designed molecules which showed good anti-inflammatory activity in ovalbumin (OVA) induced asthma in rats. The synthesis, in vitro and in vivo anti-inflammatory activity of this novel class of compounds are described.     

Splenectomy exacerbates lung injury after ischemic acute kidney injury in mice

Patients with acute kidney injury (AKI) have increased serum proinflammatory cytokines and an increased occurrence of respiratory complications. The aim of the present study was to examine the effect of renal and extrarenal cytokine production on AKI-mediated lung injury in mice. C57Bl/6 mice underwent sham surgery, splenectomy, ischemic AKI, or ischemic AKI with splenectomy and kidney, spleen, and liver cytokine mRNA, serum cytokines, and lung injury were examined. The proinflammatory cytokines IL-6, CXCL1, IL-1β, and TNF-α were increased in the kidney, spleen, and liver within 6 h of ischemic AKI. Since splenic proinflammatory cytokines were increased, we hypothesized that splenectomy would protect against AKI-mediated lung injury. On the contrary, splenectomy with AKI resulted in increased serum IL-6 and worse lung injury as judged by increased lung capillary leak, higher lung myeloperoxidase activity, and higher lung CXCL1 vs. AKI alone. Splenectomy itself was not associated with increased serum IL-6 or lung injury vs. sham. To investigate the mechanism of the increased proinflammatory response, splenic production of the anti-inflammatory cytokine IL-10 was determined and was markedly upregulated. To confirm that splenic IL-10 downregulates the proinflammatory response of AKI, IL-10 was administered to splenectomized mice with AKI, which reduced serum IL-6 and improved lung injury. Our data demonstrate that AKI in the absence of a counter anti-inflammatory response by splenic IL-10 production results in an exuberant proinflammatory response and lung injury.     

Anti-citrullinated antibodies, radiological joint damages and their correlations with disease activity score (DAS28)

Determination of anti-citrullinated peptides (anti-CCP) specificity as a predictor of joint erosive changes, correlation between their serum level and radiological damages as well as disease activity score (DAS28). A trial has been conducted on a 211 patient sample fulfilling ACR criteria for rheumatoid arthritis (RA). There was assigned anti-CCP serum level, disease activity score by the formula for DAS28(3)-CRP and assessed radiological changes degree after Steinbrocker score. In 132 patient (62.559%) the serum anti-CCP concentration was positive for RA. Specificity of the test was 100% and sensitivity 65% (Z = 0.731, p = 0.465). There is a medium intensity correlation between variables representing anti-CCP and Steinbrocker score. Pearson's coefficient was 0.479 and Spearman's rank correlation coefficient was 0.614, i.e. statistically significant (p = 0.000). There is no statistically significant correlation between variables representing anti-CCP and DAS28(3)-CRP Anti-CCP are good RA predictor and their concentration correlate with radiological damages degree.     

Comparison of the methods for profiling glycoprotein glycans--HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study

Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.