Conformation and structure of 2-amino-8-(β-d-ribofuranosyl)imidazo [1,2-a]-s-triazin-4-one (5-aza-7-deaza guanosine), a potent antiviral nucleoside

The crystal and molecular structure of one imidazo[1,2-a]-s-triazine nucleoside and its antiviral activity are described. The crystal structure of 2-amino-8-(β-d-ribofuranosyl)imidazo-[1,2-a]-s-triazin-4-one monohydrate (C₁₀H₁₃N₅O₅·H₂O) was solved by X-ray counter data. The compound crystallizes in the monoclinic space group P2₁ with cell dimensions a = 7.353 (1), b = 6.465 (1), c = 13.701 (1) Å, β = 104.64 (1)°. The structure was solved by direct methods and refined by full matrix least-squares technique to a final value of the conventional R-factor of 0.049 using 1998 observed intensities. The orientation of the base relative to the sugar ring defined in terms of rotation about the C(1′)-N(8) glycosyl bond is anti (47.8°). The ribose moiety exhibits C(2′)-endo, ²E conformation. The conformation around C(4′)-C(5′) is gauche^?. Molecular packing is dominated by hydrogen bonds. Base stacking occurs long the b axis. 5-Aza-7-deazaguanosine has shown a marked antiviral activity in vitro against herpes simplex virus despite the fact that N(3) is effective as the hydrogen acceptor only.     

Phospholipase Dδ assists to cortical microtubule recovery after salt stress

The dynamic microtubule cytoskeleton plays fundamental roles in the growth and development of plants including regulation of their responses to environmental stress. Plants exposed to hyper-osmotic stress commonly acclimate, acquiring tolerance to variable stress levels. The underlying cellular mechanisms are largely unknown. Here, we show, for the first time, by in vivo imaging approach that linear patterns of phospholipase Dδ match the localization of microtubules in various biological systems, validating previously predicted connection between phospholipase Dδ and microtubules. Both the microtubule and linear phospholipase Dδ structures were disintegrated in a few minutes after treatment with oryzalin or salt. Moreover, by using immunofluorescence confocal microscopy of the cells in the root elongation zone of Arabidopsis, we have shown that the cortical microtubules rapidly depolymerized within 30 min of treatment with 150 or 200 mM NaCl. Within 5 h of treatment, the density of microtubule arrays was partially restored. A T-DNA insertional mutant lacking phospholipase Dδ showed poor recovery of microtubule arrays following salt exposition. The restoration of microtubules was significantly retarded as well as the rate of root growth, but roots of overexpressor GFP-PLDδ prepared in our lab, have grown slightly better compared to wild-type plants. Our results indicate that phospholipase Dδ is involved in salt stress tolerance, possibly by direct anchoring and stabilization of de novo emerging microtubules to the plasma membrane, providing novel insight into common molecular mechanism during various stress events.     

Possible apoptotic mechanism in epidermal cell acantholysis induced by pemphigus vulgaris autoimmunoglobulins

Through a still unclear mechanism, pemphigus vulgaris autoantibodies (PV-IgG) induce intra-epidermal acantholytic lesions responsible for severe to fatal skin wounding. We present evidence that PV lesions contain apoptotic keratinocytes, and that cell death is induced in the lesional tissue apparently before cell separation. These data suggest that apoptosis could be the cause of the acantholytic phenomenon. We show that PV-IgG and an antibody against Fas receptor (anti-FasR) induce lesions in vitro in a similar way, causing: (1) secretion of soluble FasL; (2) elevated cellular amounts of FasR, FasL (soluble and membranal), Bax and p53 proteins; (3) reduction in levels of cellular Bcl-2; (4) enrichment in caspase 8, and activation of caspases 1 and 3; (5) co-aggregation of FasL and FasR with caspase 8 in membranal death-inducing signaling complex (DISC). Hence, the Fas-mediated death signaling pathway seems to be involved in lesion formation. Moreover, we have shown that in skin organ cultures and in keratinocyte cultures, PV-IgG can induce caspase activation and DNA fragmentation, and caspase inhibitors can prevent the formation of PV-IgG-induced epidermal lesions. Altogether, these results suggest that PV-IgG-induced acantholysis may proceed through the death-signaling pathway. They highlight new perspectives on mechanisms of tissue damage in autoimmune diseases.     

Morphological Response of the Halophilic Fungal Genus Wallemia to High Salinity

The basidiomycetous genus Wallemia is an active inhabitant of hypersaline environments, and it has recently been described as comprising three halophilic and xerophilic species: Wallemia ichthyophaga, Wallemia muriae, and Wallemia sebi. Considering the important protective role the fungal cell wall has under fluctuating physicochemical environments, this study was focused on cell morphology changes, with particular emphasis on the structure of the cell wall, when these fungi were grown in media with low and high salinities. We compared the influence of salinity on the morphological characteristics of Wallemia spp. by light, transmission, and focused-ion-beam/scanning electron microscopy. W. ichthyophaga was the only species of this genus that was metabolically active at saturated NaCl concentrations. W. ichthyophaga grew in multicellular clumps and adapted to the high salinity with a significant increase in cell wall thickness. The other two species, W. muriae and W. sebi, also demonstrated adaptive responses to the high NaCl concentration, showing in particular an increased size of mycelial pellets at the high salinities, with an increase in cell wall thickness that was less pronounced. The comparison of all three of the Wallemia spp. supports previous findings relating to the extremely halophilic character of the phylogenetically distant W. ichthyophaga and demonstrates that, through morphological adaptations, the eukaryotic Wallemia spp. are representative of eukaryotic organisms that have successfully adapted to life in extremely saline environments.Hypersaline habitats had long been considered to be populated almost exclusively by prokaryotic organisms and the research on hypersaline environments had consequently been monopolized by bacteriologists. In 2000, the first reports appeared showing that fungi are active inhabitants of solar salterns (20). Until then, fungi able to survive in environments with a low amount of biologically available water (low water activity [a_w]) were only known as contaminants of foods preserved with high concentrations of salt or sugar. Since their first discovery in salterns, many new species have been discovered in natural hypersaline environments around the world, including some species that were previously known only as food-borne contaminants. Due to these discoveries, fungi are now recognized as an integral part of indigenous halophilic microbial communities since they can grow and adjust across the whole salinity range, from freshwater to almost saturated NaCl solutions (49). Most fungi differ from the majority of halophilic prokaryotes (16): they tend to be extremely halotolerant rather than halophilic and do not require salt to remain viable, with the exception of Wallemia spp.The order Wallemiales (Wallemiomycetes, Basidiomycota) was only recently introduced to define the single genus Wallemia, a phylogenetic maverick in the Basidiomycota (49). Until 2005, this genus contained only the species W. sebi. However, taxonomic analyses of isolates from sweet, salty, and dried foods (41) and from hypersaline evaporation ponds in the Mediterranean Sea, the Caribbean, and the Dead Sea (45, 49) have resolved this genus into three species: W. ichthyophaga, W. muriae, and W. sebi. The first two of these three Wallemia spp. require additional solutes in the growth media, and W. ichthyophaga is the most halophilic eukaryote described to date, since it cannot grow without the addition of 9% NaCl (wt/vol), and it still shows growth at a_w of 0.77, equivalent to 30% NaCl (wt/vol) (49).The survival, and especially the growth, of microorganisms in highly saline environments requires numerous adaptations (6, 18, 21, 34). The dominant representatives and the most thoroughly investigated halophilic fungi in hypersaline waters of the salterns are the black yeasts, and particularly the model organism Hortaea werneckii (20). An important level of adaptation of the black yeasts to high salinity is seen in their extremophilic ecotype, which is characterized by a special meristematic morphology and changes in cell wall structure and pigmentation (27). Other fungal osmoadaptations include the accumulation of osmolytes (27, 28, 40), the extrusion of sodium (5), modification of the plasma membrane (44) and the cell wall, and even changes in fungal colony morphology (27).The fungal cell wall is the first line of defense against environmental stress; therefore, adaptation at the cell wall level is expected to have one of the most important roles for successful growth at a low a_w (24, 32). The cell wall is essential for maintaining the osmotic homeostasis of cells, since it protects them against mechanical damage as well as high concentrations of salts (7). The central fibrillar glycan network of the cell wall is embedded in highly flexible amorphous cement, which allows considerable stretching with changing osmotic pressure (14, 29). Its balance between a rigid and a dynamic structure influences the shape of cells (14) and enables growth and hyphal branching (11).Since the species within the genus Wallemia have been recognized only recently (49), little is known about their mechanisms of adaptation to high salinity. To investigate the effects of low and high NaCl concentrations on cell morphology, with particular emphasis on cell wall ultrastructure, we compared W. ichthyophaga, the most halophilic fungal species known thus far, with the related xerophilic W. muriae and W. sebi. Micrographs were prepared by using light, transmission, and scanning electron microscopy. The results reveal how this eukaryotic genus uses adaptations at the cell wall level for thriving in extremely saline environments.     

Plasma profiling reveals human fibulin-1 as candidate marker for renal impairment

There is a need for reliable and sensitive biomarkers for renal impairments to detect early signs of kidney toxicity and to monitor progression of disease. Here, antibody suspension bead arrays were applied to profile plasma samples from patients with four types of kidney disorders: glomerulonephritis, diabetic nephropathy, obstructive uropathy, and analgesic abuse. In total, 200 clinical renal-associated cases and control plasma samples from different cohorts were profiled. Parallel plasma protein profiles were obtained using biotinylated and nonfractionated samples and a selected set of 94 proteins targeted by 129 antigen-purified polyclonal antibodies. Out of the analyzed target proteins, human fibulin-1 was detected at significantly higher levels in the glomerulonephritis patient group compared to the controls and with elevated levels in patient samples for all other renal disorders investigated. Two polyclonal antibodies and one monoclonal antibody directed toward separate, nonoverlapping epitopes showed the same trend in the discovery cohorts. A technical verification using Western blot analysis of selected patient plasma confirmed the trends toward higher abundance of the target protein in disease samples. Furthermore, a verification study was carried out in the context of glomerulonephritis using an independent case and control cohort, and this confirmed the results from the discovery cohort, suggesting that plasma levels of fibulin-1 could serve as a potential indicator to monitor kidney malfunction or kidney damage.     

A new DNA marker (D4S90) is located terminally on the short arm of chromosome 4, close to the Huntington disease gene

Genetic linkage studies have mapped Huntington's disease (HD) to the distal portion of the short arm of chromosome 4 (4p16.3), 4 cM distal to D4S10 (G8). To date, no definite flanking marker has been identified. A new DNA marker, D4S90 (D5), which maps to the distal region of 4p16.3, is described. The marker was used in a genetic linkage study in the CEPH reference families with seven other markers at 4p16. The study, together with knowledge of the physical map of the region, places D4S90 as the most distal marker, 6 cM from D4S10. A provisional linkage study with HD gave a maximum lod score of 2.14 at a θ of 0.00 and no evidence of linkage disequilibrium. As D4S90 appears to be located terminally, it should play an important role in the accurate mapping and cloning of the HD gene.     

Effects of different detachment procedures on viability,nitroxide reduction kinetics and plasma membrane heterogeneity of V‐79 cells

Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V‐79 cells in the plateau phase of growth (arrested in G₁). We have measured cell viability by a dye‐exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin‐probe MeFASL(10,3) (5‐doxylpalmitoyl‐methylester), which partitions mainly in cell membranes and the hydrophilic spin‐probe TEMPONE (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin‐probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin‐treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V‐79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.     

Effects of bovine milk lactoperoxidase system on some bacteria

Bovine lactoperoxidase (LPO) was purified from skimmed milk using amberlite CG-50-H⁺ resin, CM sephadex C-50 ion-exchange chromatography, and sephadex G-100 gel filtration chromatography. Lactoperoxidase was purified 20.45-fold with a yield of 28.8%. Purity of enzyme checked by sodium dodecyl sulphate-polyacrylamide gel electrophoresis method and a single band was observed. K _m was 0.25 mM at 20°C, V _max value was 7.95 μmol/ml min at 20°C (pH 6.0). Antibacterial study was done by disk diffusion method of Kirby-Bauer using Mueller-Hinton agar medium with slight modification. Bovine LPO showed high antibacterial activity in 100 mM thiocyanate-100 mM H₂O₂ medium for some bacteria (Brevibacillus centrosaurus, B. choshinensis, B. lyticum, Cedecea davisae, Chryseobacterium indoltheticum, Clavibacter michiganense pv. insidiosum, Kocuria erythromyxa, K. kristinae, K. rosea, K. varians, Paenibacillus validus, Pseudomonas syringae pv. populans, Ralstonia pickettii, Rhodococcus wratislaviensis, Serratia fonticola, Streptomyces violaceusniger, Vibrio cholerae-nonO1) respectively, and compared with well known antibacterial substances (levofloxacin, netilmicin). LPO system has inhibition effects on all type bacteria and concentration is really important such as LPO-100 mM thiocyanate-100 mM H₂O₂ system was proposed as an effective agent against many factors causing several diseases.     

Life Cycle Assessment of Water Supply Plans in Mediterranean Spain

Life cycle assessment (LCA) was used to compare the current water supply planning in Mediterranean Spain, the so‐called AGUA Programme, with its predecessor, the Ebro river water transfer (ERWT). Whereas the ERWT was based on a single interbasin transfer, the AGUA Programme excludes new transfers and focuses instead on different types of resources, including seawater and brackish water desalination and wastewater reuse, among others. The study includes not only water supply but the whole anthropic cycle of water, from water abstraction to wastewater treatment. In addition to standard LCA impact categories, a specific impact category focusing on freshwater resources is included, which takes into account freshwater scarcity in the affected water catchments. In most impact categories the AGUA Programme obtains similar or even lower impact scores than ERWT. Concerning impacts on freshwater resources, the AGUA Programme obtains an impact score 49% lower than the ERWT. Although the current water planning appears to perform better in many impact categories than its predecessor, this study shows that water supply in Spanish Mediterranean regions is substantially increasing its energy intensity and that Mediterranean basins suffer a very high level of water stress due to increasing demand and limited resources.