The primary attachment to polymer surfaces followed by accumulation in multilayered cell clusters leads to biofilm production of Staphylococcus epidermidis, which is thought to contribute to virulence in biomaterialrelated infections. We purified a specific polysaccharide antigen of biofilm-producing S. epidermidis 1457 and RP62A, which was recently shown to have a function in the accumulative phase of biofilm production by mediating intercellular adhesion (D. Mack, M. Nedelmann, A. Krokotsch, A. Schwarzkopf, J. Heesemann, and R. Laufs, Infect. Immun. 62:3244-3253, 1994). Following Sephadex G-200 gel filtration, this antigen was separated by Q-Sepharose chromatography into a major polysaccharide, polysaccharide I (>80%), which did not bind to Q-Sepharose, and a minor polysaccharide, polysaccharide II (<20%), which was moderately anionic. As shown by chemical analyses and nuclear magnetic resonance spectroscopy, polysaccharide I is a linear homoglycan of at least 130 -1,6-linked 2-deoxy-2-amino-D-glucopyranosyl residues. On average, 80 to 85% of them are N acetylated; the rest are non-N-acetylating and positively charged. Chain cleavage by deamination with HNO 2 revealed a more or less random distribution of the non-N-acetylated glucosaminyl residues, with some prevalence of glucosaminyl-rich sequences. Cation-exchange chromatography separated molecular species whose content of non-N-acetylated glucosaminyl residues varied between 2 and 26%. Polysaccharide II is structurally related to polysaccharide I but has a lower content of non-N-acetylated Dglucosaminyl residues and contains phosphate and ester-linked succinate, rendering it anionic. Enzyme-linked immunosorbent assay inhibition with various monosaccharides revealed the -anomeric form and the acetylated amino group of the D-glucosaminyl residues as important for reactivity with the specific antiserum. The unbranched polysaccharide structure favors long-range contacts and interactions between polysaccharide strands and the cell wall and/or lectin-like proteins, leading to intercellular adhesion and biofilm accumulation. The structure of the polysaccharide is, so far, considered to be unique and, according to its function, is referred to as S. epidermidis polysaccharide intercellular adhesin (PIA).At present, coagulase-negative staphylococci, mostly Staphylococcus epidermidis, represent the most frequent cause by far of nosocomial sepsis and are the most prominent organisms responsible for infections associated with implanted biomaterials like intravascular catheters, peritoneal dialysis catheters, cerebrospinal fluid shunts, prosthetic heart valves, and prosthetic joints, resulting in substantial morbidity and mortality (25,37,54,56).By scanning electron microscopy, coagulase-negative staphylococci were shown to colonize intravascular catheters in large adherent biofilms composed of multilayered cell clusters embedded in an amorphous extracellular material, which is composed of exopolysaccharides referred to as slime or glycocalyx (13,16,28,45,51). In vitro...
We present a new method to measure absolute diffusion coefficients at nanomolar concentrations with high precision. Based on a modified fluorescence correlation spectroscopy (FCS)-setup, this method is improved by introducing an external ruler for measuring the diffusion time by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by a new two-parameter model to describe the molecule detection function (MDF). We present a recorded MDF and show the excellent agreement with the fitting model. We measure the diffusion coefficient of the red fluorescent dye Atto655 under various conditions and compare these values with a value achieved by gradient pulsed field NMR (GPF NMR). From these measurements we conclude, that the new measurement scheme is robust against optical and photophysical artefacts which are inherent to standard FCS. With two-focus-FCS, the diffusion coefficient of 4.26 x 10(-6) cm2s(-1) for Atto655 in water at 25 degrees C compares well with the GPF NMR value of 4.28 x 10(-6) cm2s(-1).
Teichoic acid (C polysaccharide) was extracted and purified from Streptococcus pneumoniae R6 with standard procedures except that lipoteichoic acid was extracted first. The dephosphorylated repeating unit was isolated after hydrolysis with 48% (by mass) HF, the bis(phosphocho1ine)-containing repeating unit was isolated by alkali hydrolysis, anion-exchange chromatography and phosphomonoester cleavage. On the basis of compositional analysis, fast-atom-bombardment mass spectrometry and NMR spectroscopy the following structure is proposed : . These observations suggested that in vivo lipoteichoic acid might regulate the activity or cellular distribution of the autolytic enzyme. For the interaction with the autolytic enzyme, the phosphocholine residues of both polymers are essential and cannot be replaced by phosphoethanolamine [7, 111. Owing to their ubiquitous occurrence among pneumococci, teichoic acid and lipoteichoic acid are pneumococcal common antigens, which suggested that antibodies against
For many applications there is a requirement for nondestructive analytical investigation of the elemental distribution in a sample. With the improvement of X-ray optics and spectroscopic X-ray imagers, full field X-ray fluorescence (FF-XRF) methods are feasible. A new device for high-resolution X-ray imaging, an energy and spatial resolving X-ray camera, is presented. The basic idea behind this so-called "color X-ray camera" (CXC) is to combine an energy dispersive array detector for X-rays, in this case a pnCCD, with polycapillary optics. Imaging is achieved using multiframe recording of the energy and the point of impact of single photons. The camera was tested using a laboratory 30 μm microfocus X-ray tube and synchrotron radiation from BESSY II at the BAMline facility. These experiments demonstrate the suitability of the camera for X-ray fluorescence analytics. The camera simultaneously records 69,696 spectra with an energy resolution of 152 eV for manganese K(α) with a spatial resolution of 50 μm over an imaging area of 12.7 × 12.7 mm(2). It is sensitive to photons in the energy region between 3 and 40 keV, limited by a 50 μm beryllium window, and the sensitive thickness of 450 μm of the chip. Online preview of the sample is possible as the software updates the sums of the counts for certain energy channel ranges during the measurement and displays 2-D false-color maps as well as spectra of selected regions. The complete data cube of 264 × 264 spectra is saved for further qualitative and quantitative processing.
Understanding the deactivation mechanism of 2-deoxy-d-ribose-5-phosphate aldolase by its natural substrate leads to a single mutant showing complete acetaldehyde resistance.
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