Biofouling on the walls of a spent nuclear fuel pool with radioactive ultrapure water

Microbial activity in spent nuclear fuel pools which contain ultrapure and radioactive water has been previously observed. The aim of the present research was to isolate and identify the microorganisms attached to the nuclear pool wall of a Spanish nuclear power plant. Amplification of 16S rDNA fragments from the culturable microorganisms by PCR using universal primers for the domain 'Bacteria', followed by Denaturing Gradient Gel Electrophoresis analysis revealed the presence of six different bacteria. The complete gene for 16S rDNA of each one was sequenced and identified as belonging to three different phylogenetic groups, viz. beta-Proteobacteria, Actinomycetales and the Bacillus/Staphylococcus group. A fungus was also found and identified as Aspergillus fumigatus by sequencing the D2 region of the large subunit rDNA gene. The isolation of these microorganisms in oligotrophic and radioactive conditions is of great interest due to the possibility of their use in bioremediation processes of radionuclide-contaminated environments.     

Analytical product study of germanium-containing medicine by different ICP-MS applications.

For several years organo-germanium containing medicine has been used for special treatments of e.g. cancer and AIDS. The active substances contain germanium as beta-carboxyethylgermanium sesquioxide ((GeCH2CH2COOH)203/"Ge-132"), spirogermanium, germanium-lactate-citrate or unspecified forms. For humans, germanium is not essential and in general the toxicity of the mentioned organo-germanium compounds is low. Acute and chronic toxic effects of inorganic germanium dioxide have been demonstrated. It is obvious that especially inorganic germanium has a higher potential of negative effects. Therefore, a widespread analytical product control is indispensable. Inductively coupled plasma mass spectrometry (ICP-MS) is the preferred technique and different applications were developed for controlling various parameters: (i) A speciation method using high performance liquid chromatography (HPLC) coupled with quadrupole (Q-) ICP-MS was developed for the identification of organo-germanium species in medicine. (ii) The nuclear magnetic resonance (NMR) technique was applied to confirm the molecular structure and to determine the molecule concentration. (iii) The total concentration of germanium in the medicine was determined in the diluted sample by high resolution (HR-) ICP-MS. (iv) For a general overview, a multi-element screening method of 56 elements with HR-ICP-MS was developed. The semi-quantitative mode was used for quantification and elements of higher abundance are reported. (v) Investigations about matrix-based interferences on masses of isotopes, which are generally determinable without remarkable problems. Isotopes like e.g. 85Rb, 88Sr, 89y, 90Zr, 93Nb and the isotopes of Ba are strongly interfered by different Ge-based molecules and need to be analysed in a higher resolution mode than used for other common matrices.     

EzrA prevents aberrant cell division by modulating assembly of the cytoskeletal protein FtsZ

In response to a cell cycle signal, the cytoskeletal protein FtsZ assembles into a ring structure that establishes the location of the division site and serves as a framework for assembly of the division machinery. A battery of factors control FtsZ assembly to ensure that the ring forms in the correct position and at the precise time. EzrA, a negative regulator of FtsZ ring formation, is important for ensuring that the ring forms only once per cell cycle and that cytokinesis is restricted to mid-cell. EzrA is distributed throughout the plasma membrane and localizes to the ring in an FtsZ-dependent manner, suggesting that it interacts directly with FtsZ to modulate assembly. We have performed a series of experiments examining the interaction between EzrA and FtsZ. As little as twofold overexpression of EzrA blocks FtsZ ring formation in a sensitized genetic background, consistent with its predicted function. A purified EzrA fusion protein interacts directly with FtsZ to block assembly in vitro. Although EzrA is able to inhibit FtsZ assembly, it is unable to disassemble preformed polymers. These data support a model in which EzrA interacts directly with FtsZ at the plasma membrane to prevent polymerization and aberrant FtsZ ring formation.     

A small, cysteine-rich protein secreted by Fusarium oxysporum during colonization of xylem vessels is required for I-3-mediated resistance in tomato

A 12 kDa cysteine-rich protein is secreted by Fusarium oxysporum f. sp. lycopersici during colonization of tomato xylem vessels. Peptide sequences obtained with mass spectrometry allowed identification of the coding sequence. The gene encodes a 32 kDa protein, designated Six1 for secreted in xylem 1. The central part of Six1 corresponds to the 12 kDa protein found in xylem sap of infected plants. A mutant that had gained virulence on a tomato line with the I-3 resistance gene was found to have lost the SIX1 gene along with neighbouring sequences. Transformation of this mutant with SIX1 restored avirulence on the I-3 line. Conversely, deletion of the SIX1 gene in a wild-type strain results in breaking of I-3-mediated resistance. These results suggest that I-3-mediated resistance is based on recognition of Six1 secreted in xylem vessels.     

Plasma levels and redox status of coenzyme Q10 in infants and children

INTRODUCTION: Increased attention has been paid to the role of lipophilic antioxidants in childhood nutrition and diseases during recent years. The lipophilic antioxidant coenzyme Q10 (CoQ10) is known as an effective inhibitor of oxidative damage. In contrast to other lipophilic antioxidants like alpha-tocopherol the plasma concentrations of CoQ10 in childhood are poorly researched. The aim of this study was to determine plasma level and redox status (oxidized form in total CoQ10 in %) of CoQ10 in clinically healthy infants, preschoolers and school-aged children. METHODS: Plasma level and redox status of CoQ10 were measured by HPLC in 199 clinically healthy children, three groups of infants [1st-4th month (n = 35), 5th-8th month (n = 25), 9th-12th month (n = 25) ], preschoolers (n = 60) and school-aged children (n = 54). The CoQ10 plasma levels were related to plasma cholesterol concentrations. The median and the 5th and 95th percentile were calculated. RESULTS: Plasma levels and redox status of CoQ10 in infants were significantly higher than in preschoolers and school-aged children. The CoQ10 redox status in the 1st-4th month was significantly increased when compared to the remaining subgroups of infants. In elder children the CoQ10 redox status stabilized. CONCLUSIONS: This is the first study concerning age-related values of plasma level and redox status of CoQ10 in apparently healthy children. Decreased CoQ10 values could be involved in various pathological conditions affecting childhood. Therefore, the application of age-adjusted reference values may provide more specific criteria to define threshold values for CoQ10 deficiency in plasma.     

Single-molecule spectroscopic methods

Being praised for the mere fact of enabling the detection of individual fluorophores a dozen years ago, single-molecule techniques nowadays represent standard methods for the elucidation of the structural rearrangements of biologically relevant macromolecules. Single-molecule-sensitive techniques, such as fluorescence correlation spectroscopy, allow real-time access to a multitude of molecular parameters (e.g. diffusion coefficients, concentration and molecular interactions). As a result of various recent advances, this technique shows promise even for intracellular applications. Fluorescence imaging can reveal the spatial localization of fluorophores on nanometer length scales, whereas fluorescence resonance energy transfer supports a wide range of different applications, including real-time monitoring of conformational rearrangements (as in protein folding). Still in their infancy, single-molecule spectroscopic methods thus provide unprecedented insights into basic molecular mechanisms.     

Studies on gangliosides with affinity for Helicobacter pylori: binding to natural and chemically modified structures

Helicobacter pylori, like many other microbes, has the ability to bind to carbohydrate epitopes. Several sugar sequences have been reported as active for the bacterium, including some neutral, sulfated, and sialylated structures. We investigated structural requirements for the sialic acid-dependent binding using a number of natural and chemically modified gangliosides. We have chosen for derivatization studies two kinds of binding-active glycolipids, the simple ganglioside S-3PG (Neu5Ac alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, sialylparagloboside) and branched polyglycosylceramides (PGCs) of human origin. The modifications included oxidation of the sialic acid glycerol chain, reduction of the carboxyl group, amidation of the carboxyl group, and lactonization. Binding experiments confirmed a preference of H. pylori for 3-linked sialic acid and penultimate 4-linked galactose. As expected, neolacto gangliosides (with Gal beta 4GlcNAc in the core structure) were active in our assays, whereas gangliosides with lacto (Gal beta 3GlcNAc) and ganglio (Gal beta 3GalNAc) carbohydrate chains were not. Negative binding results were also obtained for disialylparagloboside (with terminal NeuAc alpha 8NeuAc) and NeuAc alpha 6-containing glycolipids. Chemical studies revealed dependence of the binding on Neu5Ac and its glycerol and carboxyl side chains. Most of the derivatizations performed on these groups abolished the binding; however, some of the amide forms turned out to be active, and one of them (octadecylamide) was found to be an excellent binder. The combined data from molecular dynamics simulations indicate that the binding-active configuration of the terminal disaccharide of S-3PG is with the sialic acid in the anticlinal conformation, whereas in branched PGCs the same structural element most likely assumes the synclinal presentation.     

MprF-mediated biosynthesis of lysylphosphatidylglycerol, an important determinant in staphylococcal defensin resistance

Frequently bacteria are exposed to membrane-damaging cationic antimicrobial molecules (CAMs) produced by the host's immune system (defensins, cathelicidins) or by competing microorganisms (bacteriocins). Staphylococcus aureus achieves CAM resistance by modifying anionic phosphatidylglycerol with positively charged L-lysine, resulting in repulsion of the peptides. Inactivation of the novel S. aureus gene, mprF, which is found in many bacterial pathogens, has resulted in the loss of lysylphosphatidylglycerol (L-PG), increased inactivation by CAM-containing neutrophils, and attenuated virulence. We demonstrate here that expression of mprF is sufficient to confer L-PG production in Escherichia coli, which indicates that MprF represents the L-PG synthase. L-PG biosynthesis was studied in vitro and found to be dependent on phosphatidylglycerol and lysyl-tRNA, two putative substrate molecules. Further addition of cadaverin, a competitive inhibitor of the lysyl-tRNA synthetases, or of RNase A abolished L-PG biosynthesis, thereby confirming the involvement of lysyl-tRNA. This study forms the basis for further detailed analyses of L-PG biosynthesis and its role in bacterial infections.     

Sesquiterpenes of the liverwort Scapania undulata

The essential oil of the liverwort Scapania undulata, collected in the Harz mountains, Northern Germany, was analysed by gas chromatography (GC), GC-mass spectrometry (MS) and several new components were isolated and investigated by various NMR techniques. As new natural compounds the sesquiterpene hydrocarbons (+)-helminthogermacrene (1) [the 4Z-isomer of germacrene A (9)], (-)-cis-beta-elemene (2) as a Cope-rearrangement product of 1, (+)-beta-isolongibornene (3) and (-)-perfora-1,7-diene (4) could be identified. 1 has an identical mass spectrum and identical GC retention time on a non-polar stationary phase as germacrene A (9) but is considerably more stable than the latter. The Cope-rearrangement of 1 proceeds slowly at 350 degrees C and (-)-cis-beta-elemene (2) is formed together with small amounts of other diastereoisomers.