A high-power laser-driven source of sub-nanosecond soft X-ray pulses for single-shot radiobiology experiments

A large-scale, double-stream gas puff target has been illuminated by sub-kJ, near-infrared (NIR) focused laser pulses at the PALS facility (Prague Asterix Laser System) to produce high-energy pulses of soft X rays from hot, dense plasma. The double-puff arrangement ensures high gas density and conversion efficiency from NIR to X rays approaching that typical for solid targets. In addition, its major advantage over solid targets is that it is free of debris and has substantially suppressed charged-particle emission. The X-ray emission characteristics of the source were determined for a range of gases that included krypton, xenon, N(2), CO and N(2)-CO. A demonstrated application of the xenon-based source is a single-shot damage induction to plasmid DNA. The yields of single-strand breaks (SSBs) and double-strand breaks (DSBs) were determined as a function of energy fluence adjusted by varying distance of sample from the source and thickness of aluminum filters.     

The structure of the O-polysaccharide isolated from the lipopolysaccharide of Salmonella Dakar (serogroup O:28)

The structure of the O-antigenic part of the lipopolysaccharide (LPS) of Salmonella Dakar was analysed using chemical methods, NMR spectroscopy and mass spectrometry. The following structure for the repeating unit of the O-polysaccharide was determined: [see text] where Quip3NAc is 3-acetamido-3,6-dideoxyglucose. This is the first published structure of the O-polysaccharides from 101 serotypes of Salmonella bacteria belonging to serogroup O:28 (formerly M) in the Kauffmann-White scheme.     

The influence of vitamin D deficiency on cancers and autoimmune diseases development

There is a growing number of diseases which prevalence can be associated with vitamin D deficiency. The link between low cholecalciferol concentration and bone diseases is well established, however there is also data suggesting that it may influence development and progression of different cancers and autoimmune diseases. The in vitro studies proved that the active vitamin D metabolite--1,25(OH)(2)D(3) may arrest the cell cycle progression, induce apoptosis as well as regulate T cells and antigen presenting cells function. Results of the in vivo experiments suggest that vitamin D deficiency accelerates development of autoimmune diseases and cancers in animals. Epidemiological studies imply that the vitamin D deficiency is also associated with the increased incidence of autoimmune diseases and cancers in people. The main determinant of vitamin D serum concentration in a human body is skin synthesis. The changes in the lifestyle, air pollution as well as a common use of sun screens caused that the contemporary European receives little sunlight compared to his ancestors. According to the recent epidemiological studies, the vitamin D concentrations in serum of people who live in high latitudes (above 34 degrees N/S), including Poland, is far from being sufficient. This paper reviews results of the recent studies concerning the potential role of the vitamin D in the development of cancers and autoimmune diseases, as well as provides guidelines for vitamin D supplementation.     

Glucocorticoid-induced osteoporosis

Prolonged glucocorticoids administration is the most common cause of secondary osteoporosis. It is estimated that 30% to 50% of chronic glucocorticoids users experience vertebral or hip fractures. The highest bone loss (up to 30% in some studies) is observed in the first six months of treatment. Only a minority of patients who take chronic glucocorticoids receive optimal osteoporosis diagnosis, prevention, and/or treatment. The aim of this paper is to present the pathophysiology of glucocorticoid-induced osteoporosis, as well as some guidelines on diagnostic, preventive and therapeutic strategies for this disorder in an effort to promote the greater awareness of it.     

Phylogenetic analysis of haloalkane dehalogenases

Haloalkane dehalogenases (HLDs) are enzymes that catalyze the cleavage of carbon-halogen bonds by a hydrolytic mechanism. Although comparative biochemical analyses have been published, no classification system has been proposed for HLDs, to date, that reconciles their phylogenetic and functional relationships. In the study presented here, we have analyzed all sequences and structures of genuine HLDs and their homologs detectable by database searches. Phylogenetic analyses revealed that the HLD family can be divided into three subfamilies denoted HLD-I, HLD-II, and HLD-III, of which HLD-I and HLD-III are predicted to be sister-groups. A mismatch between the HLD protein tree and the tree of species, as well as the presence of more than one HLD gene in a few genomes, suggest that horizontal gene transfers, and perhaps also multiple gene duplications and losses have been involved in the evolution of this family. Most of the biochemically characterized HLDs are found in the HLD-II subfamily. The dehalogenating activity of two members of the newly identified HLD-III subfamily has only recently been confirmed, in a study motivated by this phylogenetic analysis. A novel type of the catalytic pentad (Asp-His-Asp+Asn-Trp) was predicted for members of the HLD-III subfamily. Calculation of the evolutionary rates and lineage-specific innovations revealed a common conserved core as well as a set of residues that characterizes each HLD subfamily. The N-terminal part of the cap domain is one of the most variable regions within the whole family as well as within individual subfamilies, and serves as a preferential site for the location of relatively long insertions. The highest variability of discrete sites was observed among residues that are structural components of the access channels. Mutations at these sites modify the anatomy of the channels, which are important for the exchange of ligands between the buried active site and the bulk solvent, thus creating a structural basis for the molecular evolution of new substrate specificities. Our analysis sheds light on the evolutionary history of HLDs and provides a structural framework for designing enzymes with new specificities.     

Regulation of human skin pigmentation and responses to ultraviolet radiation

Pigmentation of human skin is closely involved in protection against environmental stresses, in particular exposure to ultraviolet (UV) radiation. It is well known that darker skin is significantly more resistant to the damaging effects of UV, such as photocarcinogenesis and photoaging, than is lighter skin. Constitutive skin pigmentation depends on the amount of melanin and its distribution in that tissue. Melanin is significantly photoprotective and epidermal cells in darker skin incur less DNA damage than do those in lighter skin. This review summarizes current understanding of the regulation of constitutive human skin pigmentation and responses to UV radiation, with emphasis on physiological factors that influence those processes. Further research is needed to characterize the role of skin pigmentation to reduce photocarcinogenesis and to develop effective strategies to minimize such risks.     

H2AX foci in late S/G2- and M-phase cells after hydroxyurea- and aphidicolin-induced DNA replication stress in Vicia

Immunocytochemistry using α-phospho-H2AX antibodies shows that hydroxyurea (HU), an inhibitor of ribonucleotide reductase, and aphidicolin (APH), an inhibitor of DNA-polymerases α and δ, may promote formation of phospho-H2AX foci in late S/G2-phase cells in root meristems of Vicia faba. Although fluorescent foci spread throughout the whole area of nucleoplasm, large phospho-H2AX aggregates in HU-treated cells allocate mainly in perinucleolar regions. A strong tendency of ATR/ATM-dependent phospho-Chk1^S317 kinase to focus in analogous compartments, as opposed to phospho-Chk2^T68 and to both effector kinases in APH-treated cells, may suggest that selected elements of the intra-S-phase cell cycle checkpoints share overlapping locations with DNA repair factors known to concentrate in phospho-H2AX aggregates. APH-induced phosphorylation of H2AX exhibits little or no overlap with the areas positioned close to nucleoli. Following G2-M transition of the HU- and APH-pretreated cells, altered chromatin structures are still discernible as large phospho-H2AX foci in the vicinity of chromosomes. Both in HU- and APH-treated roots, immunofluorescence analysis revealed a dominant fraction of small foci and a less frequent population of large phospho-H2AX agregates, similar to those observed in animal cells exposed to ionizing radiation. The extent of H2AX phosphorylation has been found considerably reduced in root meristem cells treated with HU and caffeine. The frequencies of phospho-H2AX foci observed during mitosis and caffeine-mediated premature chromosome condensation (PCC) suggest that there may be functional links between the checkpoint mechanisms that control genome integrity and those activities which operate throughout the unperturbed mitosis in plants.     

Human lysosomal DNase IIalpha contains two requisite PLD-signature (HxK) motifs: evidence for a pseudodimeric structure of the active enzyme species

Lysosomal DNase IIalpha is essential for DNA waste removal and auxiliary apoptotic DNA fragmentation in higher eukaryotes. Despite the key role of this enzyme, little is known about its structure-function relationships. Here, mutational and biochemical analyses were used to characterize human DNase IIalpha variants expressed in mammalian cells. The resulting data strongly support the hypothesis that the enzyme is a monomeric phospholipase D-family member with a pseudodimeric protein fold. According to our results, DNase IIalpha contains two requisite PLD-signature motifs ((113)HTK(115) and (295)HSK(297)) in the N- and C-terminal subdomains, respectively, that together form a single active site. Based on these data, we present an experimentally validated structural model of DNase IIalpha.     

Increased production of laccase by <Emphasis Type="Italic">Cerrena unicolor</Emphasis> in submerged liquid cultures

The wood-degrading basidiomycete Cerrena unicolor C-139 has been suggested as a potential producer of the industrially important enzyme laccase. Basic culture parameters influencing the enzyme synthesis in shaken-flask and aerated bioreactor cultures were evaluated to improve the yields of the process. Production of extracellular laccase was considerably enhanced by the addition of Cu²⁺ in the micromolar range to a carbon-sufficient and nitrogen-sufficient culture medium (C/N = 16.69). When an optimised medium containing glucose (10 g/L) and l-asparagine (1.5 g/L) was used, and enzyme synthesis was stimulated by addition of 10 μM Cu²⁺ to the culture medium on days 3, 6 and 9, maximal laccase productivity obtained after 17 days’ cultivation in shaken flask cultures was above 100,000 nkat/L. In fermenter fungal cultures, the influence of stabilisation of medium pH on laccase activity was additionally studied. The use of a bioreactor with an automatic pH control set at pH 6.5 after 48-h incubation resulted in the enzyme activity of 65,000 nkat/L after 8 days’ cultivation.