We analyzed the emergence of daptomycin nonsusceptibility in a patient with persistent vancomycin-intermediate Staphylococcus aureus (VISA) bacteremia. The daptomycin-nonsusceptible VISA's cell wall demonstrated a reduction in muramic acid O-acetylation, a phenotypic parameter not previously reported for VISA; some isolates also contained a single point mutation in the mprF gene.

In staphylococci, crosslinking of the peptide moiety of peptidoglycan is mediated via an additional spacer, the interpeptide bridge, consisting of five glycine residues. The femAB operon, coding for two approximately 50-kDa proteins is known to be involved in pentaglycine bridge formation. Using chemical mutagenesis of the beta-lactam-resistant strain BB270 and genetic, biochemical, and biophysical characterization of mutants selected for loss of beta-lactam resistance and reduced lysostaphin sensitivity it is shown that peptide bridge formation proceeds via three intermediate bridge lengths (cell wall peptides with no, one, three, and five glycine units). To proceed from one intermediate to the next, three genes appear necessary: femX, femA, and femB. The drastic loss of beta-lactam resistance after inactivation of FemA or partial impairment of FemX even beyond the level of the sensitive wild-type strains renders these proteins attractive antistaphylococcal targets.

Determinations of the absorbed dose in a 170 MeV proton beam have been performed using seven ionization chambers of different types: five cylindrical (two FWT IC-18 and three NE-2571, of which one was modified to have the central electrode made of graphite) and two plane parallel (NACP-02 and Roos FK-6). The ionization was converted into absorbed dose in the proton beam according to the generalization of the formalism provided by the IAEA Code of Practice (TRS 277), which enables the use of the same equations for all kinds of beam used in radiotherapy. The absorbed dose obtained with the two IC-18 chambers, a chamber type commonly used as a reference in proton beams, was up to 1.5% lower than that obtained with the Farmer NE-2571 chamber, which was used as the reference in this work when calibration factors in terms of NK were used. To investigate this difference, experimental ND factors for six chambers (the two IC-18 chambers, the NACP-02, the FK-6 and two of the NE-2571 chambers) were determined in a high-energy electron beam. The procedure commonly recommended for plane parallel ion chambers was used for all the chambers, using the same reference chamber, a Farmer NE-2571. In the 170 MeV proton beam all the ND factors yielded consistent absorbed dose determinations within the estimated experimental uncertainties. This finding calls into question the value of the product kattkm for the IC-18 chamber given by the IAEA Code of Practice used in this comparison, and points at possible chamber to chamber variations that theoretical kattkm factors cannot predict. The investigations enabled the determination of the Pwall(60Co) factor of the Roos FK-6 plane parallel chamber, yielding 1.003 +/- 0.5%, and a correction for the effect of the aluminium central electrode of NE-2571 chambers in proton beams, equal to 1.003 +/- 0.4%. Two of the chambers (the plane parallel FK-6 and the modified cylindrical NE-2571) were provided with calibration factors in terms of absorbed dose to water, Nw, at the quality of 60Co by the Primary Standard Dosimetry Laboratory in Germany (PTB). Using the Nw formalism excellent agreement was found with the determination based on the experimental ND, giving support to the implementation of the NW procedure in therapeutic proton beams.

Quantitative distance measurements are difficult to obtain in spite of the strong distance dependency of the energy transfer efficiency. One problem for the interpretation of the Forster resonant energy transfer (FRET) efficiency is the so-called zero-efficiency peak caused by FRET pairs with missing or nonfluorescent acceptors. Other problems occurring are direct excitation of the acceptor, spectral crosstalk, and the determination of the quantum efficiency of the dyes as well as the detector sensitivity. Our approach to overcome these limitations is based on the pulsed-interleaved excitation (PIE) of both the acceptor and the donor molecule. PIE is used to excite the acceptor dye independently of the FRET process and to prove its existence via fluorescence. This technique enables us to differentiate a FRET molecule, even with a very low FRET efficiency, from a molecule with an absent or non-fluorescent acceptor. Crosstalk, direct acceptor excitation, and molecular brightness of acceptor and donor molecules are determined by analyzing the data with fluorescence correlation spectroscopy (FCS). FRET efficiencies of the same data set are also determined by analyzing the lifetimes of the donor fluorophores. The advantages of the PIE-FRET approach are demonstrated on a polyproline assay labeled with Alexa-555 and Alexa-647 as donor and acceptor, respectively.

A new mild experimental approach for isolation of peptide membrane receptors and subsequent analysis of post-translational modifications is described. Endothelin receptors A and B were isolated on oligo(dT)-cellulose using N-(⑀-maleimidocaproyloxy)succinimide endothelin coupled to a protected (dA)-30-mer. This allowed a one-step isolation of the receptor from oligo(dT)-cellulose via variation solely of salt concentration. The identity of the receptor was confirmed by direct amino acid sequencing of electroblotted samples or by using antibodies against ET A and ET B receptors. Although it is clear that post-translational modifications of G-protein-coupled receptors are intimately involved in the physiological function of these signal transduction systems, there is as yet relatively little direct evidence for the specific modifications and the relationship of the different modifications to signal transduction processes. These types of processes are not easily amenable to analysis by genomic methods, i.e. there is a need for efficient methods to directly analyze these processes at the proteome level. We report here new, efficient methods for rapid isolation of the endothelin B receptor and for highly sensitive analysis of its post-translational modifications via mass spectrometry.Endothelin, the strongest vasoconstrictor yet known, is a 21-amino acid peptide with physiological effects on cellular development, differentiation, vasoconstriction, and mitogenesis (1, 2). There are 3 different endothelin isoforms, ET-1, 1 ET-2, and ET-3 with different affinity for the two different endothelin receptor subtypes, A and B (3-6). Both receptors are members of the G-protein-coupled receptor superfamily (3-6, 7). The consensus ET receptor topology includes three extracellular domains, three intracellular loops, and a cytoplasmic COOH-terminal tail, separated by seven hydrophobic helical regions thought to span the lipid bilayer. In addition it has been presumed that ET receptors are post-translationally modified by glycosylation of the NH 2 terminus and by phosphorylation and palmitoylation of the cytoplasmic surface. Based on homology with other G-protein-coupled receptors (8 -10), there has been speculation regarding possible structures, functional regions, and sites of post-translational modifications for endothelin receptors (11-16). However, as yet there is very little direct evidence for attributes such as the sites and the roles of glycosylation, palmitoylation, and phosphorylation, the location of the endothelin-binding site and the basis for discrimination among the three different endothelin isoforms.A role of endothelin in disease has recently been demonstrated by the finding that mutated ET B receptor is associated with Hirschsprung's disease (17-21). In addition, mice lacking the ET-1 gene display severe malformation of large blood vessels, stressing the importance of endothelin during development (22). Endothelin has been shown to be a mitogenic agonist in different cell types (23, 24). The signaling pathway by ...

Rat bradykinin B 2 receptor from unstimulated Chinese hamster ovary cells transfected with the corresponding cDNA has been isolated, and subsequent mass spectrometric analysis of multiple phosphorylated species and of the palmitoylation attachment site is described. Bradykinin B 2 receptor was isolated on oligo(dT)-cellulose using N-(⑀-maleimidocaproyloxy)succinimide-Met-Lys-bradykinin coupled to a protected (dA) 30 -mer. This allowed a one-step isolation of the receptor on an oligo(dT)-cellulose column via variation solely of salt concentration. After enzymatic in-gel digestion, matrix-assisted laser desorption ionization and electrospray ion trap mass spectrometric analysis of the isolated rat bradykinin B 2 receptor showed phosphorylation at Bradykinin, a member of the kinin family (1), is a nonapeptide with diverse biological activities ranging from a role in the inflammatory process to regulatory effects on vascular permeability, blood pressure, renal homeostasis, and pain generation (2, 3). Bradykinin mediates its physiological effects by binding to and activation of the bradykinin B 2 receptor. Molecular cloning has revealed the primary structure of the B 2 receptor (4) and classified it as a member of the G protein-coupled receptor superfamily. The consensus bradykinin receptor topology predicts four extracellular domains (ED 1 1-4) and intracellular domains (ID1-4), each separated by seven transmembrane helical regions (TM1-7) spanning the lipid bilayer. B 2 receptors are post-translationally modified by glycosylation (5), phosphorylation (6), and presumably by palmitoylation of the cytoplasmic surface.Based on homology with other G protein-coupled receptors there have been indications regarding possible structural features probed by agonists, antagonists, and anti-idiotypic antibodies (5, 6), functional regions (7), and sites of post-transitional modifications for the B 2 receptor (8). Site-directed mutagenesis indicated the importance of Tyr residues and of ID4 for the signaling and the uptake of the B 2 receptor in receptor in rat-1 cells transfected with wild and mutant receptor cDNAs (9). However, as yet there is still rather little direct evidence at the protein level for attributes such as the precise sites and the roles of glycosylation, palmitoylation, and phosphorylation of bradykinin B 2 or other G protein-coupled receptors. Indeed phosphorylation sites for few G protein-coupled receptors have been mapped to date (10 -12), and most of these sites reflect the in vitro phosphorylation of the isolated receptor.We report here on the isolation of rat B 2 receptor from transfected Chinese hamster ovary (CHO) cells using oligo(dA) covalently linked to bradykinin via a specially developed bifunctional cross-linker. Affinity chromatography has been carried out under very mild conditions using oligo(dT) columns analogous to methods used for isolation of eukaryotic mRNA. In-gel digestion of electrophoretically separated receptor, subsequent peptide mass fingerprinting by matrix-assisted laser desorption...

R ecently, a variety of protein transduction domains (PTDs) have been identified allowing the transfer of peptides, proteins, or nucleic acids across cellular membranes into cells. 1-9 PTDs have been used to successfully treat preclinical models of human disease such as cancer, psoriasis, and stroke. 10-13 Transfer of nano-particular structures across cellular membranes is of increasing importance for the development of novel diagnostic and therapeutic tools. The capacity of PTDs to mediate an endocytosis-independent transfer of particles across the plasma membrane into the cytoplasm is still unclear. To investigate the potential of PTD to mediate the transfer of nano-particles across the cell membrane, virus-like particles (VLPs) harboring a marker gene were instrumental.Hepatitis B virus (HBV) core particles represent a very well-characterized VLP model system. 14,15 The HBV core particle (capsid) is assembled by 180 or 240 core protein monomers (HBcAg), resulting in an icosahedral particle 30 or 34 nm in diameter, respectively. A characteristic of the core protein is a basic arginine-rich C-terminal region, which is responsible for the packaging of DNA 16 and for guidance of the capsid to the nucleus. 17 Based on extensive structural analysis, 18 the hepatitis B capsid was used as a carrier for foreign epitopes to develop new vaccines. 19,20 In the viral context, the core particle harbors the viral genome (nucleocapsid). Efficient in vivo packaging of nucleic acids into capsids requires HBV polymerase and its interaction with a defined secondary structure (termed epsilon) at the 5Ј end of the RNA to be packaged. 21 The ⑀ signal is the primary element of the hepadnaviral packag-

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