A Versatile High-Vacuum Cryo-transfer System for Cryo-microscopy and Analytics.

Cryogenic microscopy methods have gained increasing popularity, as they offer an unaltered view on the architecture of biological specimens. As a prerequisite, samples must be handled under cryogenic conditions below their recrystallization temperature, and contamination during sample transfer and handling must be prevented. We present a high-vacuum cryo-transfer system that streamlines the entire handling of frozen-hydrated samples from the vitrification process to low temperature imaging for scanning transmission electron microscopy and transmission electron microscopy.

Daptomycin: local application in implant-associated infection and complicated osteomyelitis.

Background: The rise of highly resistant bacteria creates a persistent urge to develop new antimicrobial agents. This paper investigates the application of the lipopeptide antibiotic daptomycin in infections involving the human bone.

Methods: Compressive and tensile strength testing of daptomycin-laden PMMA was performed referring to the ISO 5833.

High-resolution investigation of nanoparticle interaction with a model pulmonary surfactant monolayer.

The pulmonary surfactant film spanning the inner alveolar surface prevents alveolar collapse during the end-exhalation and reduces the work of breathing. Nanoparticles (NPs) present in the atmosphere or nanocarriers targeted through the pulmonary route for medical purposes challenge this biological barrier. During interaction with or passage of NPs through the alveolar surfactant, the biophysical functioning of the film may be altered.

Differential cytotoxic actions of Shiga toxin 1 and Shiga toxin 2 on microvascular and macrovascular endothelial cells.

Shiga toxin (Stx)-mediated injury to vascular endothelial cells in the kidneys, brain and other organs underlies the pathogenesis of haemolytic uraemic syndrome (HUS) caused by enterohaemorrhagic Escherichia coli (EHEC). We present a direct and comprehensive comparison of cellular injury induced by the two major Stx types, Stx1 and Stx2, in human brain microvascular endothelial cells (HBMECs) and EA.hy 926 macrovascular endothelial cells.

Heterologous expression of Anabaena sp. PCC7120 cyanophycin metabolism genes cphA1 and cphB1 in Sinorhizobium (Ensifer) meliloti 1021.

Sinorhizobium meliloti infects leguminous plants resulting in a nitrogen-fixing symbiosis. Free living cells accumulate poly(3-hydroxybutyrate) (PHB) as carbon and energy source under imbalanced growth conditions. The cphA1 (7120) gene encoding a cyanophycin (CGP) synthetase of Anabaena sp.

Characterization of rubber particles and rubber chain elongation in Taraxacum koksaghyz.

Background: Natural rubber is a biopolymer with exceptional qualities that cannot be completely replaced using synthetic alternatives. Although several key enzymes in the rubber biosynthetic pathway have been isolated, mainly from plants such as Hevea brasiliensis, Ficus spec. and the desert shrub Parthenium argentatum, there have been no in planta functional studies, e.

Analysis of lipid export in hydrocarbonoclastic bacteria of the genus Alcanivorax: identification of lipid export-negative mutants of Alcanivorax borkumensis SK2 and Alcanivorax jadensis T9.

Triacylglycerols (TAGs), wax esters (WEs), and polyhydroxyalkanoates (PHAs) are the major hydrophobic compounds synthesized in bacteria and deposited as cytoplasmic inclusion bodies when cells are cultivated under imbalanced growth conditions. The intracellular occurrence of these compounds causes high costs for downstream processing. Alcanivorax species are able to produce extracellular lipids when the cells are cultivated on hexadecane or pyruvate as the sole carbon source.

Establishment of a novel anabolism-based addiction system with an artificially introduced mevalonate pathway: complete stabilization of plasmids as universal application in white biotechnology.

Plasmid stability in recombinant microorganisms is a very important requirement for highly efficient plasmid-based production processes in biotechnology. To stably maintain plasmids, we developed in this study an efficient and stringent novel anabolism-based addiction system, which can be widely used. This novel addiction system is based on two components: (i) an Escherichia coli HMS174(DE3) knockout mutant of the ispH gene coding for 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC 1.

Electron tomography of structures in the wall of hazel pollen grains.

The three-dimensional structure of channels and bacula cavities in the wall of hazel pollen grains was investigated by automated electron tomography in order to explore their role in the release of allergen proteins from the pollen grains. 3D reconstructions of 100-150 nm thick resin-embedded sections, stabilized by thin platinum-carbon coating, revealed that the channels aimed directly towards the surface of the grain and that the bacula cavities were randomly sized and merged into larger ensembles. The number and the dimensions of the ensembles were quantitatively determined by neighboring voxel analysis on thresholded reconstructed volumes.

Alpha/beta hydrolase 1 is upregulated in D5 dopamine receptor knockout mice and reduces O2- production of NADPH oxidase.

Renal dopamine receptors have been shown to play a critical role in ROS-dependent hypertension. D5 dopamine receptor deficient (D5-/-) mice are hypertensive and have increased systemic oxidative stress which is manifested in the kidney and the brain. To further investigate the underlying mechanisms of hypertension in D5-/- mice, we used RNA arrays to compare mRNA levels of kidneys from wildtype and D5-/- mice.

MASDET-A fast and user-friendly multiplatform software for mass determination by dark-field electron microscopy.

Electron microscopy has been used to measure the mass of biological nanoparticles since the early 60s, and is the only way to obtain the mass of large structures or parameters such as the mass-per-length of filaments. The ability of this method to sort heterogeneous samples both in terms of mass and shape promises to make it a key tool for proteomics down to the single cell level. A new multiplatform software package, MASDET, that can be run under MATLAB or as a standalone program is described.

Damages of the tibial post in constrained total knee prostheses in the early postoperative course - a scanning electron microscopic study of polyethylene inlays.

Background: Investigation of the risk of fracture of the polyethylene (PE) inlay in constrained total knee prostheses.

Methods: Three unused and seven polyethylene inlays that had been implanted in a patient's knee for an average of 25.4 months (min 1.

Ralstonia eutropha H16 flagellation changes according to nutrient supply and state of poly(3-hydroxybutyrate) accumulation.

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE), in combination with matrix-assisted laser desorption ionization-time of flight analysis, and the recently revealed genome sequence of Ralstonia eutropha H16 were employed to detect and identify proteins that are differentially expressed during different phases of poly(3-hydroxybutyric acid) (PHB) metabolism. For this, a modified protein extraction protocol applicable to PHB-harboring cells was developed to enable 2D PAGE-based proteome analysis of such cells. Subsequently, samples from (i) the exponential growth phase, (ii) the stationary growth phase permissive for PHB biosynthesis, and (iii) a phase permissive for PHB mobilization were analyzed.

Synthesis and accumulation of cyanophycin in transgenic strains of Saccharomyces cerevisiae.

Cyanophycin [multi-L-arginyl-poly(L-aspartic acid) (CGP)] was, for the first time, produced in yeast. As yeasts are very important production organisms in biotechnology, it was determined if CGP can be produced in two different strains of Saccharomyces cerevisiae. The episomal vector systems pESC (with the galactose-inducible promoter GAL1) and pYEX-BX (with the copper ion-inducible promoter CUP1) were chosen to express the cyanophycin synthetase gene from the cyanobacterium Synechocystis sp.

Bundles of hexagonally arranged tubules in timothy grass pollen: detection of a novel pollen component using anhydrous fixation and image analysis techniques in transmission electron microscopy.

Pollen from timothy grass (Phleum pratense L.) was subjected to various aqueous and non-aqueous fixation and preparation protocols for transmission electron microscopy. Only in the cytoplasm of anhydrously prepared pollen grains were conspicuous inclusions observed that range in size from less than 1 mum up to 8 or 10 mum.

Identification and localization of cyanophycin in bacteria cells via imaging of the nitrogen distribution using energy-filtering transmission electron microscopy.

In this study the technique of energy-filtering transmission electron microscopy was applied to localize cyanophycin (CGP) in recombinant strains of Ralstonia eutropha. Since CGP is a polymer consisting of the amino acids aspartate and arginine, which functions as a temporary nitrogen reserve that is deposited as insoluble inclusions in the cytoplasm of the cell, its nitrogen content is significantly higher than that of the other cell matter. In this study, we recorded nitrogen distribution maps, which represent the location of CGP in ultrathin sections of resin-embedded cells of recombinant strains of R.

Heat-shock protein HspA mimics the function of phasins sensu stricto in recombinant strains of Escherichia coli accumulating polythioesters or polyhydroxyalkanoates.

Polyhydroxyalkanoic acids (PHAs) are synthesized by unspecific PHA synthases and deposited as energy and carbon storage granules in the cytoplasm of many prokaryotes. The number and size of the granules depend on the presence of phasins which are amphiphilic structural proteins occurring at the granule surface. Recently, it was shown that polythioesters (PTEs) are also synthesized by PHA synthases.

Analysis of storage lipid accumulation in Alcanivorax borkumensis: Evidence for alternative triacylglycerol biosynthesis routes in bacteria.

Marine hydrocarbonoclastic bacteria, like Alcanivorax borkumensis, play a globally important role in bioremediation of petroleum oil contamination in marine ecosystems. Accumulation of storage lipids, serving as endogenous carbon and energy sources during starvation periods, might be a potential adaptation mechanism for coping with nutrient limitation, which is a frequent stress factor challenging those bacteria in their natural marine habitats. Here we report on the analysis of storage lipid biosynthesis in A.

Calcium-dependent tetramer formation of S100A8 and S100A9 is essential for biological activity.

S100 proteins comprise the largest family of calcium-binding proteins. Members of this family usually form homo- or heterodimers, which may associate to higher-order oligomers in a calcium-dependent manner. The heterodimers of S100A8 and S100A9 represent the major calcium-binding proteins in phagocytes.

Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters.

Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price.

Electron beam irradiation of poly(vinyl methyl ether) films: 1. Synthesis and film topography.

Temperature-sensitive hydrogel layers on silicon (Si) substrates were synthesized by electron beam irradiation of spin-coated poly(vinyl methyl ether) (PVME) films. The influences of the used solvent, the polymer concentration, and the spinning velocity on the homogeneity and the thickness of the PVME film were investigated. In the range of concentration c(p) = 1-15 wt% PVME in ethanol solution, homogeneous films with a thickness between d = 50 nm and 1.

Group 13 allergens as environmental and immunological markers for grass pollen allergy: studies by immunogold field emission scanning and transmission electron microscopy.

Background: Polygalacturonases were recently identified as important grass pollen allergens and designated group 13 allergens. The objective of the present study was to investigate the presence of group 13 grass pollen allergens in different grass species, their release and ultrastructural location in dry and hydrated grass pollen.

Methods: Nitrocellulose-blotted allergen extracts from 12 wild and cultivated grass genera were probed with a rabbit antiserum raised against purified recombinant timothy grass pollen allergen, Phl p 13.

Physiological conditions conducive to high cyanophycin content in biomass of Acinetobacter calcoaceticus strain ADP1.

The effects of the inorganic medium components, the initial pH, the incubation temperature, the oxygen supply, the carbon-to-nitrogen ratio, and chloramphenicol on the synthesis of cyanophycin (CGP) by Acinetobacter calcoaceticus strain ADP1 were studied in a mineral salts medium containing sodium glutamate and ammonium sulfate as carbon and nitrogen sources, respectively. Variation of all these factors resulted in maximum CGP contents of only about 3.5% (wt/wt) of the cell dry matter (CDM), and phosphate depletion triggered CGP accumulation most substantially.

The wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase.

The wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) catalyzes the final steps in triacylglycerol (TAG) and wax ester (WE) biosynthesis in the gram-negative bacterium Acinetobacter sp. strain ADP1. It constitutes a novel class of acyltransferases which is fundamentally different from acyltransferases involved in TAG and WE synthesis in eukaryotes.

Mechanism of lipid-body formation in prokaryotes: how bacteria fatten up.

Neutral lipid accumulation is frequently observed in some Gram-negative prokaryotes like Acinetobacter sp. and most actinomycetes, including the pathogenic Mycobacterium tuberculosis and antibiotic producing streptomycetes. We examined the formation of wax ester- and triacylglycerol (TAG)-bodies in Acinetobacter calcoaceticus and Rhodococcus opacus using microscopic, immunological and biophysical methods.