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15546755 | Molecular-orientation analysis based on alignment-induced TROSY chemical shift changes. | We present a new NMR technique for determining the alignment tensor of a weakly aligned protein using only alignment-induced 15N transverse relaxation optimized spectroscopy (TROSY) chemical shift changes. Alignment-induced TROSY chemical shift changes reflect the combined contributions from two different anisotropic spin interactions including the residual dipolar couplings (RDCs) and the residual chemical shift anisotropy effects (RCSAs). We show here that these two residual anisotropic spin interactions' values, encoded in the TROSY chemical shift changes, can be used to determine a weakly aligned protein's alignment tensor. To prove the significance of this method, we show that our TROSY-based analysis gives the consistent alignment angles with those determined using RDCs for 15N-labeled ubiquitin (8.6 kDa) in an aligned medium, within an uncertainty range estimated by considering experimental and structural noises, being 5 degrees at most. Because our approach requires a pre-determined 15N CSA tensor value, we also estimated the uncertainties associated with the resultant alignment tensor values caused by variation in 15N CSA tensors. In spite of the significant variations in literature-reported 15N CSA tensors, they gave consistent orientation angles within an uncertainty range. These results ensure that our TROSY-based approach is a useful alternative to the RDC-based method to determine the alignment angles especially for large proteins in a weakly aligned state. |
15546754 | Covariance NMR spectroscopy by singular value decomposition. | Covariance NMR is demonstrated for homonuclear 2D NMR data collected using the hypercomplex and TPPI methods. Absorption mode 2D spectra are obtained by application of the square-root operation to the covariance matrices. The resulting spectra closely resemble the 2D Fourier transformation spectra, except that they are fully symmetric with the spectral resolution along both dimensions determined by the favorable resolution achievable along omega2. An efficient method is introduced for the calculation of the square root of the covariance spectrum by applying a singular value decomposition (SVD) directly to the mixed time-frequency domain data matrix. Applications are shown for 2D NOESY and 2QF-COSY data sets and computational benchmarks are given for data matrix dimensions typically encountered in practice. The SVD implementation makes covariance NMR amenable to routine applications. |
15546753 | Sensitivity-enhanced double-TROSY experiment for simultaneous measurement of one-bond 15N-1H, 15N-13C' and two-bond 1H-13C' couplings. | A recently published experiment for the measurement of 1JHN, 1JNC', and 2JHC' coupling constants [J. Am. Chem. Soc. 125 (2003) 11504] was modified to yield a double-TROSY experiment which selects 1 of the 16 multiplet components from a 15N-HSQC spectrum recorded of a uniformly 15N/13C-labelled protein. Subspectra containing any 1 of the 16 multiplet components can be generated allowing accurate coupling constant measurements. The experiment is sensitivity enhanced, turning all magnetization components precessing during the evolution time into observable magnetization during the detection time. The experiment is discussed with regard to the previously published alpha/beta-filtered HN(alpha/beta-NC'-J) experiment [J. Magn. Reson. 140 (1999), 32] which measures the same coupling constants. |
15546752 | Weak alignment of membrane proteins in stressed polyacrylamide gels. | Residual dipolar couplings are important as angular constraints for the structure determination of membrane proteins in micelles. Strained polyacrylamide gels are one of the few available mechanisms available for inducing the requisite weak alignment for these samples. However, their use is frequently limited by the ability to incorporate proteins and buffer solutions into the gel matrix. The implementation of several methods of incorporating membrane proteins into gels are described. Conditions for copolymerizing the protein in the absence of a change in pH are detailed. Electrophoresis is also shown to be a useful method to incorporate proteins. Weak alignment of the protein-micelle complex in the gel matrix is subsequently achieved using either vertical or radial compression. The magnitude of alignment can be controlled by altering the gel concentration, the acrylamide/bisacrylamide ratio, and the compression ratio. The alignment tensor can be altered relative to uncharged polyacrylamide gels by copolymerizing samples with acrylamide/acrylic acid to incorporate negative charges in the strained polyacrylamide gel to provide an alternate orientation. |
15546751 | Magnetic relaxation dispersion probe. | The magnetic field dependence of nuclear spin-lattice relaxation rates provides a powerful approach to characterizing intra and intermolecular dynamics. NMR spectrometers that provide extensive magnetic relaxation dispersion profiles may switch magnetic field strengths rapidly by either moving the sample or by changing the current in an electromagnet. If the sample is moved, the polarization and detection fields may be very high, which provides both high sensitivity and resolution. This report summarizes the design of a pneumatic sample transport system for glass sample containers that may be used in either a dual magnet spectrometer or in a single magnet system that exploits the fringe field as the secondary magnetic field. |
15546750 | Separation and characterization of different signals from intermolecular three-spin orders in solution NMR. | In this paper, signals originating from a pure specific coherence of intermolecular three-spin orders were separated and characterized experimentally in highly polarized two-component spin systems. A modified CRAZED sequence with selective radio-frequency excitation was designed to separate the small signals from the strong conventional single-spin single-quantum signals. General theoretical expressions of the pulse sequence with arbitrary flip angle pulses were derived using dipolar field treatment. The expressions were used to predict the relaxation and diffusion properties and optimal experimental parameters such as flip angles. For the first time, relaxation and diffusion properties of pure intermolecular single-quantum, double-quantum, and triple-quantum coherences of three-spin orders were characterized and analyzed in one-dimensional experiments. All experimental observations are in excellent agreement with the theoretical predictions. The theoretical results show that the quantum-mechanical treatment leads to exactly the same predictions as the dipolar field treatment. The quantitative study of intermolecular multiple-quantum coherences of three-spin orders presented herein provides a better understanding of their mechanisms. |
15546749 | Modulation of the distance dependence of paramagnetic relaxation enhancements by CSA x DSA cross-correlation. | Paramagnetic metal ions with fast-relaxing electronic spin and anisotropic susceptibility tensor provide a rich source of structural information that can be derived from pseudo-contact shifts, residual dipolar couplings, dipole-dipole Curie spin cross-correlation, and paramagnetic relaxation enhancements. The present study draws attention to a cross-correlation effect between nuclear relaxation due to anisotropic chemical shielding (CSA) and due to the anisotropic dipolar shielding (DSA) caused by the electronic Curie spin. This CSA x DSA cross-correlation contribution seems to have been overlooked in previous interpretations of paramagnetic relaxation enhancements. It is shown to be sufficiently large to compromise the 1/r6 distance dependence usually assumed. The effect cannot experimentally be separated from auto-correlated DSA relaxation. It can increase or decrease the observed paramagnetic relaxation enhancement. Under certain conditions, the effect can dominate the entire paramagnetic relaxation, resulting in nuclear resonances narrower than in the absence of the paramagnetic center. CSAxDSA cross-correlation becomes important when paramagnetic relaxation is predominantly due to the Curie rather than the Solomon mechanism. Therefore the effect is most pronounced for relaxation by metal ions with large magnetic susceptibility and fast-relaxing electron spin. It most strongly affects paramagnetic enhancements of transverse relaxation in macromolecules and of longitudinal relaxation in small molecules. |
15546748 | An NMR study of the origin of dioxygen-induced spin-lattice relaxation enhancement and chemical shift perturbation. | Due to its depth-dependent solubility, oxygen exerts paramagnetic effects which become progressively greater toward the hydrophobic interior of micelles, and lipid bilayer membranes. This paramagnetic gradient, which is manifested as contact shift perturbations (19F and 13C NMR) and spin-lattice relaxation enhancement (19F and 1H NMR), has been shown to be useful for precisely determining immersion depth, membrane protein secondary structure, and overall topology of membrane proteins. We have investigated the influence of oxygen on 19F and 13C NMR spectra and spin-lattice relaxation rates of a semiperfluorinated detergent, (8,8,8)-trifluoro (3,3,4,4,5,5,6,6,7,7)-difluoro octylmaltoside (TFOM) in a model membrane system, to determine the dominant paramagnetic spin-lattice relaxation and shift-perturbation mechanism. Based on the ratio of paramagnetic spin-lattice relaxation rates of 19F and directly bonded 13C nuclei, we conclude that the dominant relaxation mechanism must be dipolar. Furthermore, the temperature dependence of oxygen-induced chemical shift perturbations in 9F NMR spectra suggests a contact interaction is the dominant shift mechanism. The respective hyperfine coupling constants for 19F and 13C nuclei can then be estimated from the contact shifts <(deltav/v0)19F> and <(deltav/v0)13C>, allowing us to estimate the relative contribution of scalar and dipolar relaxation to 19F and 13C nuclei. We conclude that the contribution to spin-lattice relaxation from the oxygen induced paramagnetic scalar mechanism is negligible. |
15546747 | Molecular dynamics and information on possible sites of interaction of intramyocellular metabolites in vivo from resolved dipolar couplings in localized 1H NMR spectra. | Proton NMR resonances of the endogenous metabolites creatine and phosphocreatine ((P)Cr), taurine (Tau), and carnosine (Cs, beta-alanyl-L-histidine) were studied with regard to residual dipolar couplings and molecular mobility. We present an analysis of the direct 1H-1H interaction that provides information on motional reorientation of subgroups in these molecules in vivo. For this purpose, localized 1H NMR experiments were performed on m. gastrocnemius of healthy volunteers using a 1.5-T clinical whole-body MR scanner. We evaluated the observable dipolar coupling strength SD0 (S=order parameter) of the (P)Cr-methyl triplet and the Tau-methylene doublet by means of the apparent line splitting. These were compared to the dipolar coupling strength of the (P)Cr-methylene doublet. In contrast to the aliphatic protons of (P)Cr and Tau, the aromatic H2 (delta=8 ppm) and H4 (delta=7 ppm) protons of the imidazole ring of Cs exhibit second-order spectra at 1.5 T. This effect is the consequence of incomplete transition from Zeeman to Paschen-Back regime and allows a determination of SD0 from H2 and H4 of Cs as an alternative to evaluating the multiplet splitting which can be measured directly in high-resolution 1H NMR spectra. Experimental data showed striking differences in the mobility of the metabolites when the dipolar coupling constant D0 (calculated with the internuclear distance known from molecular geometry in the case of complete absence of molecular dynamics and motion) is used for comparison. The aliphatic signals involve very small order parameters S approximately (1.4-3) x 10(-4) indicating rapid reorientation of the corresponding subgroups in these metabolites. In contrast, analysis of the Cs resonances yielded S approximately (113-137) x 10(-4). Thus, the immobilization of the Cs imidazole ring owing to an anisotropic cellular substructure in human m. gastrocnemius is much more effective than for (P)Cr and Tau subgroups. Furthermore, 1H NMR experiments on aqueous model solutions of histidine and N-acetyl-L-aspartate (NAA) enabled the assignment of an additional signal component at delta=8 ppm of Cs in vivo to the amide group at the peptide bond. The visibility of this proton could result from hydrogen bonding which would agree with the anticipated stronger motional restriction of Cs. Referring to the observation that all dipolar-coupled multiplets resolved in localized in vivo 1H NMR spectra of human m. gastrocnemius collapse simultaneously when the fibre structure is tilted towards the magic angle (theta; approximately 55 degrees), a common model for molecular confinement in muscle tissue is proposed on the basis of an interaction of the studied metabolites with myocellular membrane phospholipids. |
15546746 | Phase modulation in dipolar-coupled A2 spin systems: effect of maximum state mixing in 1H NMR in vivo. | Coupling constants of nuclear spin systems can be determined from phase modulation of multiplet resonances. Strongly coupled systems such as citrate in prostatic tissue exhibit a more complex modulation than AX connectivities, because of substantial mixing of quantum states. An extreme limit is the coupling of n isochronous spins (An system). It is observable only for directly connected spins like the methylene protons of creatine and phosphocreatine which experience residual dipolar coupling in intact muscle tissue in vivo. We will demonstrate that phase modulation of this "pseudo-strong" system is quite simple compared to those of AB systems. Theory predicts that the spin-echo experiment yields conditions as in the case of weak interactions, in particular, the phase modulation depends linearly on the line splitting and the echo time. |
15546745 | Extending the limits of the selective 1D NOESY experiment with an improved selective TOCSY edited preparation function. | Compared to its 2D counterpart, the selective 1D NOESY experiment offers greatly simplified spectral interpretation and is invaluable to the structure elucidation of small-to-medium sized molecules, although its application is limited to well-resolved resonances only. The doubly selective 1D TOCSY-NOESY experiment allows the 1D NOESY experiment to be extended to resonances within overlapped spectral regions. However, existing methods do not address the critical issue of zero-quantum interference, which leads to severe anti-phase distortions to the line shape of scalar coupled spins and often complicates the identification of weak NOE enhancements. In this communication, we describe an improved selective TOCSY edited preparation (STEP) function and its application to the selective 1D NOESY experiment. The STEP function incorporates a novel zero-quantum filter introduced by Thrippleton and Keeler [Angew. Chem. Int. Ed. 42 (2003) 3938], which permits essentially complete suppression of zero-quantum coherence in a single scan. Residual anti-phase distortions due to spin-state mixing are removed using the double difference methodology reported by Shaka et al. [45th Experimental NMR Conference, Pacific Grove, USA, 2004]. The combined use of these techniques ensures that the final spectra are free of distortions, which is crucial to the reliable detection of weak NOE enhancements. Although employed as an additional preparation period in the example demonstrated here, the STEP function affords a general editing tool for spectral simplification and can be applied to a range of experiments. |
15546744 | Random coil phosphorus chemical shift of deoxyribonucleic acids. | Random coil phosphorus chemical shift has been studied using 16 17-nucleotide DNA sequences. Due to the presence of residual base stacking in these sequences, the temperature and sequence effects were investigated at 50 and 55 degrees C. The phosphorus chemical shifts of random coil DNA sequences have been found to be independent of temperature. Sequence effect analysis shows that the phosphorus chemical shift of a nucleotide in a random coil DNA sequence depends on both its 5'- and 3'-nearest neighbors. A trimer model has been used to establish the random coil 31P chemical shift prediction protocol which shows an accuracy of 0.02 ppm. |
15546743 | Chimeric cyclodepsipeptides as mimetics for the anthelmintic PF1022A. | In the anthelmintic cyclooctadepsipeptide PF1022A (1) didepsipeptide units have been exchanged for the beta-turn mimetics (D)-Pro-(L)-Pro and BTD (7) in order to elucidate the functional role of the depsipeptide backbone. Compounds 12 and 14 are the first PF1022A analogues in which a substantial part of the PF1022A backbone has been replaced with an improvement of anthelmintical activity. Preliminary structure-activity relationships suggest a symmetric conformation to be the biological active one. |
15546742 | Evans Blue is an inhibitor of nuclear factor-kappa B (NF-kappaB)-DNA binding. | Nuclear factor-kappa B (NF-kappaB) is an important transcription factor, involved in many immune and inflammatory responses. It is critical in HIV gene expression as it has kappa B binding sites in the HIV-1 long-terminal repeat. Hence, targeting NF-kappaB to prevent its DNA binding holds a significant therapeutic potential. In this context, we report Evans Blue as a novel inhibitor of NF-kappaB-DNA binding. Evans Blue was found to be inhibiting DNA binding of NF-kappaB at a low concentration of 100 microM. Further, molecular modeling studies using docking and generation of electrostatic potential maps predicted a possible binding mode of EB to the DNA binding region of NF-kappaB, consistent with the experimental activity. |
15546741 | 9-cis-retinoic acid analogues with bulky hydrophobic rings: new RXR-selective agonists. | Stille cross-coupling of aryltriflates 10 and dienylstannane 11, oxidation and Horner-Wadsworth-Emmons reaction afforded stereoselectively retinoates 15. Saponification provided the carboxylic acids 8a and 8b, retinoids that incorporate a bulky hydrophobic ring while preserving the 9-cis-geometry of the parent system. In contrast to the pan-RAR/RXR agonistic profile of the lower homologue of 8a, compound 7 (LG100567), retinoids 8 showed selective binding and transactivation of RXR, devoid of significant RAR activation. In PLB985 leukemia cells that require RXR agonists for differentiation compounds 8 induced maturation in the presence of the RAR-selective pan-agonist TTNPB; this effect was blocked by an RXR-selective antagonist. |
15546740 | Conversion of human-selective PPARalpha agonists to human/mouse dual agonists: a molecular modeling analysis. | To understand the species selectivity in a series of alpha-methyl-alpha-phenoxy carboxylic acid PPARalpha/gamma dual agonists (1-11), structure-based molecular modeling was carried out in the ligand binding pockets of both human and mouse PPARalpha. This study suggested that interaction of both 4-phenoxy and phenyloxazole substituents of these ligands with F272 and M279 in mouse PPARalpha leads to the species-specific divergence in ligand binding. Insights obtained in the molecular modeling studies of these key interactions resulted in the ability to convert a human-selective PPARalpha agonist to a human and mouse dual agonist within the same platform. |
15546739 | The synthesis and evaluation of [2.2.1]-bicycloazahydantoins as androgen receptor antagonists. | A novel series of [2.2.1]-azahydantoins has been designed and synthesized in an enantiospecific manner. The ability of these compounds to act as antagonists to the androgen receptor was investigated and several were found to have potent activity in vitro. |
15546738 | A comparative binding study of modified bovine immunodeficiency virus TAR RNA against its TAT peptide. | Besides generating novel binding peptides or small molecules to their RNA target, successful design of chemically modified RNA constructs capable of tighter binding with their binding peptides is also of significant importance. Herein, the synthesis and binding studies of a series of both wt and mutant bovine immunodeficiency virus (BIV) TAR RNA constructs against its Tat peptide are reported. Understanding the requirements that enable RNA construct binding properties, especially at the hairpin loop or internal bulge, would afford potential therapeutic approaches to control the BIV life cycle. |
15546737 | Aminoimidazo[1,2-a]pyridines as a new structural class of cyclin-dependent kinase inhibitors. Part 1: Design, synthesis, and biological evaluation. | We have identified a novel structural class of protein serine/threonine kinase inhibitors comprised of an aminoimidazo[1,2-a]pyridine nucleus. Compounds from this family are shown to potently inhibit cyclin-dependent kinases by competing with ATP for binding to a catalytic subunit of the protein. Structure-based design approach was used to direct this chemical scaffold toward generating potent and selective CDK2 inhibitors. The discovery of this new class of ATP-site directed protein kinase inhibitors, aminoimidazo[1,2-a]pyridines, provides the basis of new medicinal chemistry tool in search for an effective treatment of cancer and other diseases that involve protein kinase signaling pathways. |
15546736 | QSAR modelling of HIV-1 reverse transcriptase inhibition by benzoxazinones using a combination of P_VSA and pharmacophore feature descriptors. | In pursuit of better anti-HIV drugs, a quantitative structure-activity relationship analysis using a novel set of 2D descriptors was performed on a series of HIV-1 reverse transcriptase inhibitory benzoxazinones. The QSAR models derived from the above mentioned descriptors were found to be statistically significant and exhibited superior predictive power. The results of the study justify the application of the descriptors for exploring the binding mode of the benzoxazinones to the enzyme. |
15546735 | Identification of a highly potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries. | A solution-phase multiple-parallel synthesis approach was employed for the preparation of 6-, 7- and 8-aryl-substituted chromenone libraries, which were screened as inhibitors of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). These studies resulted in the identification of 8-dibenzothiophen-4-yl-2-morpholin-4-yl-chromen-4-one (NU7441) as a highly potent and selective DNA-PK inhibitor (IC50=14 nM), exhibiting ATP-competitive inhibition kinetics. |
15546734 | Alumina supported potassium permanganate: an efficient reagent for chemoselective dehydrogenation of 2-imidazolines under mild conditions. | Potassium permanganate supported on alumina was found to be an efficient reagent system for dehydrogenation of 2-imidazolines to imidazoles under mild conditions at room temperature. Selective oxidation of 2-alkylimidazolines in the presence of 2-arylimidazolines was achieved using this reagent system. 2-Imidazolines were also selectively converted to their corresponding imidazoles in the presence of other oxidizable functional groups such as sulfide, ether, aldehyde, acetal and THP ether. The oxidation procedure described here is easy to carry out and does not require strict reaction conditions. |
15546733 | Novel diarylsulfonylurea derivatives as potent antimitotic agents. | A novel series of diarylsulfonylurea derivatives were synthesized and evaluated for interaction with tubulin and for cytotoxicity against human cancer cell lines. These derivatives demonstrated good inhibitory activity against tubulin polymerization, which was well correlated with promising antiproliferative activity as well as G2/M phase cell cycle arrest. Furthermore, several compounds were also efficacious against multidrug-resistant cancer cells, which are resistant to many other known microtubule inhibitors. |
15546732 | Structure-based virtual screening and biological evaluation of potent and selective ADAM12 inhibitors. | We describe a series of potent and selective inhibitors of ADAM12 that were discovered using computational screening of a focused virtual library. The initial structure-based virtual screening selected 64 compounds from a 3D database of 67,062 molecules. Being evaluated by a cell-based ADAM12 activity assay, compounds 5, 11, 14, 16 were further identified as the potent and selective inhibitors of ADAM12 with low nanomolar IC50 values. The mechanism underlying the potency and selectivity of a representative compound, 5, was investigated through molecular docking studies. |
15546731 | Improving the intein-mediated, site-specific protein biotinylation strategies both in vitro and in vivo. | One of the critical issues in the generation of a protein microarray lies in the choice of immobilization strategies, which ensure proteins are adhered to the glass surface while properly retaining their native biological activities. We previously developed intein-mediated strategies for protein biotinylation and site-specific protein microarray generation. Herein, we report new findings of these strategies, which improve the biotinylation efficiency of proteins by up to 10-folds. |
15546730 | Discovery of novel 2-(aminoheteroaryl)-thiazole-5-carboxamides as potent and orally active Src-family kinase p56(Lck) inhibitors. | A series of substituted 2-(aminoheteroaryl)-thiazole-5-carboxamide analogs have been synthesized as novel, potent inhibitors of the Src-family kinase p56Lck. Among them, compound 2 displayed superior in vitro potency and excellent in vivo efficacy. |
15546729 | Synthesis and anticonvulsant activity of new 2-substituted-5- [2-(2-fluorophenoxy)phenyl]-1,3,4-oxadiazoles and 1,2,4-triazoles. | A series of new 2-substituted-5-[2-(2-fluorophenoxy)phenyl]-1,3,4-oxadiazoles has been synthesized and screened for their anticonvulsant activities. Compound 3 shows considerable anticonvulsant activity both in PTZ and MES models. It seems that this effect is mediated by benzodiazepine receptors and other unknown mechanism, respectively. |
15546728 | Design of bivalent ligands using hydrogen bond linkers: synthesis and evaluation of inhibitors for human beta-tryptase. | We exploit the concept of using hydrogen bonds to link multiple ligands for maintaining simultaneous interactions with polyvalent binding sites. This approach is demonstrated by the syntheses and evaluation of pseudo-bivalent ligands as potent inhibitors of human beta-tryptase. |
15546727 | 3-(2-Methoxytetrahydrofuran-2-yl)pyrazoles: a novel class of potent, selective cyclooxygenase-2 (COX-2) inhibitors. | A series of 3-(2-methoxytetrahydrofuran-2-yl)pyrazoles (4-10) was synthesized. The compounds were evaluated for their ability to inhibit cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) activity in human whole blood (HWB). The compound, 5-(4-methanesulfonylphenyl)-3-(2-methoxytetrahydrofuran-2-yl)-1-p-tolyl-1H-pyrazole 5 showed potent and selective COX-2 inhibition (IC50 for COX-1: >100 microM and COX-2: 1.2 microM). |
15546726 | Development of an efficient and selective radioligand for bradykinin B1 receptor occupancy studies. | We have developed an efficient and selective radioligand, the [35S]-radiolabeled dihydroquinoxalinone derivative, 4, for an ex vivo receptor occupancy assay in transgenic rats over-expressing the human bradykinin B1 receptor. |
15546725 | Antiviral compounds from traditional Chinese medicines Galla Chinese as inhibitors of HCV NS3 protease. | Under the guidance of bioassay, the EtOAc extract fraction of the Traditional Chinese Medicine (TCM) Galla Chinese was found to be efficient in inhibiting the NS3 protease of HCV and purified the fraction to get three polyphenol compounds 1,2,6-tri-O-galloyl-beta-D-glucose (1), 1,2,3,6-tetra-O-galloyl-beta-D-glucose (2), and 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (3), which were identified as inhibitors of Hepatitis C Virus (HCV) NS3 protease. Compounds 1, 2, and 3 inhibited HCV NS3 protease with IC50 of 1.89, 0.75, and 1.60 microM, respectively. |
15546724 | Design, synthesis and anti-microbial activity of 1H-pyrazole carboxylates. | In a SAR study, we have synthesized a few 1H-pyrazole carboxylate related microbicides using Vilsmeier reagent. The anti-microbial screening results of 1H-pyrazole-3-carboxylate are reported here for the first time. The effect of 1H-pyrazole carboxylates on the mycelial growth of plant pathogenic fungi is revealed. The first X-ray structure in the family of microbicidal 1H-pyrazole-4-carboxylates is presented. |
15546723 | Fluorescence quenching of parallel-stranded DNA bound ethidium bromide: the effect of 7-deaza-2'-deoxyisoguanosine and 7-halogenated derivatives. | Parallel stranded (ps) duplexes were constructed by incorporating 7-deaza-2'-deoxyisoguanosine (1a) or its 7-halogenated analogs 1b,c in place of 2'-deoxyisoguanosine. UV and Tm analyses prove the high affinity of ethidium bromide (EB) to these modified duplexes. Steady-state fluorescence measurement shows that the fluorescence is quenched when EB is bound to ps duplexes containing compounds 1a-c. The quenching effect depends on the 7-substituent of the nucleobase. |
15546722 | Synthesis, (1-->3)-beta-D-glucanase-binding ability and phytoalexin-elicitor activity of (R)-2,3-epoxypropyl (1-->3)-beta-D-pentaglucoside. | The (1-->3)-beta-D-pentaglucoside was synthesized as its (R)-2,3-epoxypropyl glycoside via 2+3 strategy. The disaccharide donor 8 was obtained by 3-selective coupling of 2 with 4, followed by deallylation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor 12 was prepared by coupling of 10 with 4, followed by deacetylation. Condensation of 8 with 12, followed by epoxidation, and deprotection, gave the target pentaoside. The results of these bioassays demonstrated that the (1-->3)-beta-D-glucanase was obviously inactivated by 15 with k(app)=3.79 x 10(-4) min(-1). At the same time, we found that the 15 was more active as compared to the laminaripentaose in eliciting phytoalexin accumulation in tobacco cotyledon tissue, and it could be kept longer time than laminaripentaose, which indicated it is much more stable than laminaripentaose. |
15546720 | CVT-4325: a potent fatty acid oxidation inhibitor with favorable oral bioavailability. | New inhibitors of palmitoyl-CoA oxidation are based on the introduction of nitrogen heterocycles in the 'Western Portion' of the molecule. SAR studies led to the discovery of CVT-4325 (shown), a potent FOXi (IC50=380 nM rat mitochondria) with favorable PK properties (F=93%, t(1/2)=13.6h, dog). |
15546721 | Tocopherol long chain fatty alcohols decrease the production of TNF-alpha and NO radicals by activated microglial cells. | The synthesis of a series of Tocopherol long chain Fatty Alcohols (TFA) and their biological activities on the modulation of microglial activation are described. Specifically, the 2-(12-hydroxy-dodecyl)-2,5,7,8-tetramethyl-chroman-6-ol, the TFA bearing 12 carbon atoms on the side chain (n=12), shows the most potent inhibition of secretion on nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) by lipopolysaccharide (LPS)-activated microglia. |
15546719 | Design, synthesis and evaluation of bicyclic benzamides as novel 5-HT1F receptor agonists. | Several fused bicyclic systems have been investigated to serve as the core structure of potent and selective 5-HT1F receptor agonists. Replacement of the indole nucleus in 2 with indazole and 'inverted' indazole provided more potent and selective 5-HT1F receptor ligands. Indoline and 1,2-benzisoxazole systems also provided potent 5-HT1F receptor agonists, and the 5-HT1A receptor selectivity of the indoline- and 1,2-benzisoxazole-based 5-HT1F receptor agonists could be improved with modification of the benzoyl moiety of the benzamides. Through these studies, we found that the inherent geometries of the templates, not the nature of hybridization of the linking atom, were important for the 5-HT1F receptor recognition. |
15546718 | Synthesis of 8-thiabicyclo[3.2.1]oct-2-enes and their binding affinity for the dopamine and serotonin transporters. | The reinforcing and stimulant properties of cocaine have been primarily associated with its propensity to bind to monoamine transport systems, in particular the dopamine transporter. Inhibition of the dopamine transporter then leads to an increase of synaptic dopamine with substantial pharmacological consequences. The search for medications for cocaine abuse has had a particular focus on tropane analogs of cocaine, and the interchange of nitrogen for oxygen in this class has led to potent and selective inhibitors of monoamine transport. Herein we report that 8-thiatrop-2-enes are highly potent and quite selective inhibitors of the dopamine transporter. The 3,4-dichlorophenyl-8-thiabicyclo[3.2.1]oct-2-ene (4f) is particularly potent (IC50=4.5 nM) and selective (800-fold) with respect to inhibition of the serotonin transporter. |
15546717 | Carbonic anhydrase inhibitors. Inhibition of the prokariotic beta and gamma-class enzymes from Archaea with sulfonamides. | A detailed inhibition study of carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the beta- and gamma-families from Archaea with sulfonamides has been performed. Compounds included in this study were the clinically used sulfonamide CA inhibitors, such as acetazolamide, methazolamide, ethoxzolamide, topiramate, valdecoxib, celecoxib, dorzolamide, sulfanilamide, dichlorophanamide, as well as sulfanilamide analogs, halogenated sulfanilamides, and some 1,3-benzenedisulfonamide derivatives. The two gamma-CAs from Methanosarcina thermophila (Zn-Cam and Co-Cam) showed very different inhibitory properties with these compounds, as compared to the alpha-CA isozymes hCA I, II, and IX, and the beta-CA from Methanobacterium thermoautotrophicum (Cab). The best Zn-Cam inhibitors were sulfamic acid and acetazolamide, with inhibition constants in the range of 63-96 nM, whereas other investigated aromatic/heterocylic sulfonamides showed a rather levelled behavior, with KIs in the range of 0.12-1.70 microM. The best Co-Cam inhibitors were topiramate and p-aminoethyl-benzenesulfonamide, with KIs in the range of 0.12-0.13 microM, whereas the worst one was homosulfanilamide (KI of 8.50 microM). In the case of Cab, the inhibitory power of these compounds varied to a much larger extent, with sulfamic acid and sulfamide showing millimolar affinities (KIs in the range of 44-103 mM), whereas the best inhibitor was ethoxzolamide, with a KI of 5.35 microM. Most of these sulfonamides showed inhibition constants in the range of 12-100 microM against Cab. Thus, the three CA families investigated up to now possess a very diverse affinity for sulfonamides, the inhibitors with important medicinal, and environmental applications. |
15546716 | Isoxazole-3-hydroxamic acid derivatives as peptide deformylase inhibitors and potential antibacterial agents. | A series of isoxazole-3-hydroxamic acid derivatives has been identified as a new class of small, nonpeptidic inhibitors of peptide deformylase (PDF). The synthesis, enzyme inhibition and preliminary investigation of the binding mode of this potential antibacterial compounds are reported. |
15546715 | Alpha-1-C-alkyl-1-deoxynojirimycin derivatives as potent and selective inhibitors of intestinal isomaltase: remarkable effect of the alkyl chain length on glycosidase inhibitory profile. | A series of alpha- and beta-1-C-alkyl-1-deoxynojirimycin derivatives was prepared and evaluated as glycosidase inhibitors. Biological assays showed a marked dependence of the selectivity and potency of the inhibitors upon the position of the alkyl chain (alpha-1-C-, beta-1-C- or N-alkyl derivatives). In addition, the efficiency of alpha-1-C-alkyl-1-deoxynojirimycin derivatives as intestinal isomaltase inhibitors increases with the length of the alkyl chain. The strongest inhibition was found for alpha-1-C -nonyl-1-deoxynojirimycin with an IC50=3.5 nM (25x more potent inhibitor than the shorter chain homologue carrying a C8 chain). These results demonstrate that subtle changes in the aglycon fragment may result in remarkable enzyme specificity. |
15546714 | Identification of a novel non-carbohydrate molecule that binds to the ribosomal A-site RNA. | We report the identification of a novel compound that binds to the Escherichia coli 16S ribosomal A-site. Binding by the compound was observed using nuclear magnetic resonance and mass spectrometry techniques. We show that the compound binds in the same position in the A-site RNA as occupied by the aminoglycoside class of antibiotics. |
15546713 | Synthesis and interfacial behavior of sulfur-containing analogs of lung surfactant dipalmitoyl phosphatidylcholine. | Synthesis methods and initial surface property characterizations are reported for two sulfur-containing phosphonolipids related structurally to dipalmitoyl phosphatidylcholine (DPPC), the major lung surfactant glycerophospholipid. Sulfur linkages in these compounds affect molecular interactions relative to ester linkages, and are structurally resistant to cleavage by phospholipases. The SO2-linked analog synthesized here had increased adsorption and improved film respreading compared to DPPC, while reaching very low surface tensions (1 N/m) in cycled interfacial films on both the Wilhelmy balance and the pulsating bubble surfactometer. This compound appears to have potential utility as a component in future phospholipase-resistant synthetic exogenous surfactants for treating clinical forms of inflammatory lung injury. |
15546712 | Multidrug resistance reversal activity of permethyl ningalin B amide derivatives. | A series of amide derivatives of permethyl ningalin B were prepared and examined as multidrug resistance (MDR) reversal agents illustrating that the C5 carboxylate is widely tolerant of such derivatization. |
15546711 | Isosteric N-arylpiperazine replacements in a series of dihydropyridine NPY1 receptor antagonists. | 4-Amino-N-arylpiperidines serve as effective bioisosteres for N-arylpiperazines in the series of dihydropyridine NPY1 receptor antagonists. These were prepared by a ZnCl2-mediated reductive amination reaction between elaborated primary amines, 2 or 5, and 4-arylpiperidones. |
15546710 | Development of a new class of potential antiatherosclerosis agents: NO-donor antioxidants. | A new class of NO-donor phenol derivatives is described. The products were obtained by joining appropriate phenols with either nitrooxy or 3-phenylsulfonylfuroxan-4-yloxy moieties. All the compounds proved to inhibit the ferrous salt/ascorbate induced lipidic peroxidation of membrane lipids of rat hepatocytes. They were also capable of dilating rat aorta strips precontracted with phenylephrine. |
15546709 | Synthesis and antitrypanosomal profile of new functionalized 1,3,4-thiadiazole-2-arylhydrazone derivatives, designed as non-mutagenic megazol analogues. | In this work we reported the synthesis and the trypanocidal profile of new 1,3,4-thiadiazole-2-arylhydrazone derivatives of nitroimidazole series (4) or phenyl series (5), designed by exploring the molecular hybridization approach between megazol (2) and guanyl hydrazone derivative (3). The evaluation of the activity against bloodstream trypomastigote forms of Trypanosoma cruzi forms lead us to identify a new potent trypamomicide prototype, that is, brazilizone A (4k), which present an IC50/24 h=5.3 microM. |
15546708 | Tryptamine-based human beta3-adrenergic receptor agonists. Part 2: SAR of the methylene derivatives. | A series of tryptamine derivatives with modified sulfonamide were designed, synthesized, and evaluated for their ability to stimulate cAMP accumulation in CHO cells expressing the cloned human beta3-adrenergic receptor (AR). For this series of compounds, our objective was to symmetrize the alpha-position of the tryptamine moiety maintaining its activity and reducing the cost of production. Compound 11h, having m-aminobenzene, exhibited excellent agonistic activity for beta3-AR with excellent subtype selectivity. |
15546707 | Tryptamine-based human beta3-adrenergic receptor agonists. Part 1: SAR studies of the 7-position of the indole ring. | A series of tryptamine-based 2-thiophenesulfonamide derivatives were prepared and their agonistic activity for the beta-adrenergic receptors (ARs) was evaluated. Compound 54, containing 7-methanesulfonyloxy tryptamine, was found to be a highly potent beta3-AR agonist (EC50=0.21 nM, IA=97%) with excellent selectivity for the beta3-AR over the beta1- and beta2-ARs (210- and 86-fold, respectively). |
15546706 | Relationship between performance on the Stroop test and N-acetylaspartate in the medial prefrontal cortex in deficit and nondeficit schizophrenia: preliminary results. | The aim of this research was to investigate the relationship between performance on the Stroop test and N-acetylaspartate/creatine assessed using proton magnetic resonance spectroscopy in the medial prefrontal cortex (MPFC) of schizophrenia patients. The Schedule for the Deficit Syndrome was used to subdivide the schizophrenia patients into deficit (n=5) and nondeficit (n=17) subtypes. Twenty-one control subjects served as a comparison group. A strong correlation between right-sided N-acetylaspartate/creatine levels and Stroop scores was found in the deficit patients but not in the nondeficit patients and the controls. This result suggests a relationship between a dysfunction of the right medial prefrontal cortex and a deficit in selective attention in schizophrenia patients with the deficit syndrome. |
15546705 | 3 Tesla magnetic resonance imaging of the brain in newborns. | While it has been hypothesized that brain development is abnormal in schizophrenia and other neurodevelopmental disorders, there have been few attempts to study very early brain development in children. Twenty unsedated healthy newborns underwent 3 Tesla magnetic resonance imaging (MRI), including diffusion tensor imaging (DTI). The left ventricle was significantly larger than the right; females had significantly larger ventricles than males. Fractional anisotropy (FA) increased significantly with gestational age in the genu and splenium of the corpus callosum. It is feasible to study brain development in unsedated newborns using 3 T MRI. |
15546704 | A meta-analysis of functional neuroimaging in obsessive-compulsive disorder. | Recent neurobiological models of obsessive-compulsive disorder (OCD) posit that a dysfunction in orbitofrontal-subcortical circuitry underlies the etiology of this disorder. Much of the empirical support for these theories comes from studies using neuroimaging techniques to compare brain activity in OCD patients with that in non-OCD controls. Qualitative reviews of this literature implicate the orbitofrontal cortex, caudate nuclei, and thalamus. In this study, a meta-analysis was conducted to summarize the results of studies using positron emission tomography (PET) and single photon emission computed tomography (SPECT) to investigate brain activity in OCD. Results suggest that differences in radiotracer uptake between patients with OCD and healthy controls have been found consistently in the orbital gyrus and the head of the caudate nucleus. No other significant differences were found. The implications of these results for theories regarding the etiology of OCD are discussed. |
15546703 | Validation of semiautomated methods for quantifying cingulate cortical metrics in schizophrenia. | This paper validates semiautomated methods for reconstructing cortical surfaces of the cingulate gyrus from high-resolution magnetic resonance (MR) images. Bayesian segmentation was used to delineate the image voxels into five tissue types: cerebrospinal fluid (CSF), gray matter (GM), white matter (WM), and partial volumes of CSF/GM and GM/WM; the tissues were then recalibrated as CSF, GM, and WM via the Neyman-Pearson Likelihood Ratio Test. To generate cortical surfaces at the interface of GM and WM, the thresholds between the tissue types were first used to reassign partial volume voxels to CSF, GM, and WM with minimum error (that varied from 0.06 to 0.15 for the 10 subjects). Next, topology-correct cortical surfaces were generated and validated with almost all surface vertices lying within one voxel (0.5 mm) of hand contours. Dynamic programming was used to delineate and extract the cingulate gyrus from the cortical surfaces based on its gyral and sulcal boundaries. The intraclass correlation coefficient for surface area obtained by two raters for all 10 surfaces was 0.82. In addition, by repeating the entire procedure three times in one subject, we obtained a coefficient of variation of 0.0438 for surface area. |
15546702 | Volume reduction of the amygdala in patients with schizophrenia: a magnetic resonance imaging study. | The amygdala is known to be involved in the pathology of schizophrenia. While only a limited number of studies in schizophrenia have measured the amygdala as a single structure. The aim of this study was to examine the hypothesis that patients with schizophrenia would show reduced volumes in the amygdala compared with normal controls. We investigated amygdala volume in 40 patients with schizophrenia (20 males, 20 females) and 40 age- and gender-matched normal controls using three-dimensional magnetic resonance imaging (MRI). Whole volumes of both the amygdala and the temporal lobe were measured on consecutive coronal 1-mm slices. The amygdala volume was significantly smaller in schizophrenia patients than in controls. Considering gender differences, male patients had significantly smaller volumes in the bilateral amygdala than male controls; female patients had a significantly reduced right amygdala compared with female controls. Furthermore, a significant left-smaller-than-right volumetric asymmetry of the amygdala was detected in male patients with schizophrenia. The results may be important for understanding the role of the amygdala in the pathophysiology of schizophrenia and the anatomical substrates of gender difference in the expressions of the illness. |
15546701 | Comparative study of proton and phosphorus magnetic resonance spectroscopy in schizophrenia at 4 Tesla. | This study used high-field magnetic resonance spectroscopy to examine the correlation of 1H and 31P metabolite levels in patients with schizophrenia and normal controls. 1H and 31P in vivo spectra were acquired successively from the left anterior cingulate and left thalamus of nine chronic schizophrenic patients and eight comparable healthy controls. A significant positive correlation between glutamine (Gln) and phosphoethanolamine (PEtn) was found in the left anterior cingulate of patients. In the left thalamus of patients, a significant negative correlation between N-acetylaspartate (NAA) and glycerophosphocholine (GroPCho) was found. No significant correlations were found in controls. The correlation between glutamine and phosphoethanolamine may reflect a link between neurotransmission alterations and membrane phospholipid metabolism alterations. The negative correlation between N-acetylaspartate and glycerophosphocholine may reflect the presence of neurodegeneration. |
15546700 | Differences of cerebral activation between superior and inferior learners during motor sequence encoding and retrieval. | Cerebral activation during memory encoding and retrieval might depend on subjects' learning capacity, either by corresponding to better performance in superior learners or by reflecting increased effort in inferior learners. To investigate these alternative hypotheses, the study compared cerebral activation during encoding and retrieval of a motor sequence in groups of subjects with superior and inferior learning performances. Ten healthy subjects underwent functional magnetic resonance imaging (fMRI) while performing a motor sequence encoding paradigm (i.e. finger tapping sequence) and a retrieval paradigm (i.e. reproduction of the learned sequence). Subjects were divided into superior and inferior learners according to the correctness of sequence reproduction during retrieval. During encoding, there was strong bilateral activation in the middle frontal gyrus, the supplementary motor area (SMA), the lateral parietal lobe and the cerebellum. During retrieval, again strong activation was found in identical areas of the prefrontal cortex, the parietal lobe and the cerebellum. During encoding, inferior learners showed more left-sided activations in the left middle frontal and inferior parietal gyri. Superior learners showed increased activation in the corresponding right-sided brain areas during encoding as well as during retrieval. Differences of cerebral activations in the prefrontal and parietal cortex during encoding and retrieval were found to be related to retrieval performance, i.e. success and effort. Further intervention studies are needed to assess whether these interindividual differences are the cause or the consequence of differences in memory performance. |
15546699 | A PET provocation study of generalized social phobia. | In an investigation of the neural circuits that may mediate the subjective experience of social phobia (SP), six male patients with a primary DSM-IV diagnosis of generalized social phobia watched, in the presence of a group of "communication experts," a videotape of themselves giving an impromptu talk (Exposure condition). In the control Baseline condition, they viewed a videotape of a socially competent stranger giving a talk. Regional cerebral blood flow was measured thrice under each condition. The study revealed significant deactivations from Baseline during Exposure in the right lingual gyrus (BA 18) and in the right medial frontal gyrus (BA 11); significant activations during Exposure were not observed. Deactivation of these regions may reflect a strategy of visual avoidance employed by the patients to dampen their phobic experience. |
15546698 | Reduced temporal lobe volume in early onset conduct disorder. | Regional brain volumes derived from magnetic resonance imaging (MRI) scans from 10 youths with early onset conduct disorder and 10 healthy controls matched for age, sex and handedness were compared to determine whether prefrontal or temporal lobe brain volumes differed in the two groups. Right temporal lobe and right temporal gray matter volumes were significantly reduced in subjects with conduct disorder compared with controls. Prefrontal volumes in subjects with conduct disorder were 16% smaller than in controls, but the difference did not reach statistical significance. Early onset conduct disorder without substance abuse comorbidity was also significantly associated with smaller right temporal gray volumes. Further investigation of both the temporal and frontal localizations of the pathophysiology of early onset conduct disorder is warranted in larger samples. |
15546689 | Subacute toxicity evaluation of a new camptothecin anticancer agent CKD-602 administered by intravenous injection to rats. | The subacute toxicity of a new camptothecin anticancer agent, CKD-602, was investigated after 4-week repeated intravenous administration of the chemical in Sprague-Dawley rats. The test chemical was administered intravenously to rats at dose levels of 0, 0.003, 0.013, or 0.067 mg/kg/day for males and 0, 0.004, 0.018, or 0.089 mg/kg/day for females. At the end of the treatment period, 10 rats/sex/group were sacrificed. The remaining 5 rats/sex in the vehicle control and high dose groups continued the study without treatment for 2 weeks (recovery period). During the test period, clinical signs, mortality, body weights, food and water consumption, ophthalmoscopy, urinalysis, hematology, serum biochemistry, gross findings, organ weights, and histopathology were examined. In both sexes of the high dose group, an increase in the incidence of abnormal clinical signs and paleness of the eyes, a reduction in the body weight gain, food consumption and urine protein, and an increase in the water consumption were observed. Hematological investigations revealed a decrease in the red blood cells, hemoglobin and hematocrit and an increase in the mean corpuscular volume, mean corpuscular hemoglobin, platelets, and reticulocytes in a dose-dependent manner. Serum total cholesterol and total protein values were lower in females than those of controls, but not in males. An increase in the heart and liver weights and a decrease in the thymus weight were also found. Histopathological alterations included an increase in the incidence of atrophy of the sternal marrow, atrophy, fibrosis and mast cell hyperplasia of the femoral marrow, atrophy of the white pulp and extramedullary hematopoiesis of the spleen, atrophy of the thymus, auricular hypertrophy of the heart, extramedullary hematopoiesis and centriacinar telangiectasis of the liver, follicular degeneration of the ovary, and inflammation of the tail. The major treatment-related effects were not recovered at the end of 2-week recovery period. There were no adverse effects in the low and middle dose groups of both genders. In the present experimental conditions, the target organs were determined to be bone marrow, blood cells, spleen, liver, thymus, and heart. The no-observed-adverse-effect level was considered to be 0.013 mg/kg/day for males and 0.018 mg/kg/day for females. |
15546688 | Regulatory immunotoxicology: does the published evidence support mandatory nonclinical immune function screening in drug development? | Recent immunotoxicity guidance documents from the EU CHMP and the US FDA apply significantly different weightings to immune function testing; whereas the former mandates (as a starting point) incorporation of immune function tests (IFTs) to screen for immunotoxic potential in sub-chronic rodent toxicity studies, the more cautious 'for cause' FDA approach recommends the use of IFTs only when warranted by evidence obtained from conventional nonclinical and/or clinical studies. Conclusions from detailed evaluations of several key drugs, including salmeterol and some opioids, challenge the notion that data on these examples support the need for IFTs to detect unintended immunosuppression. Given the virtual absence of convincing pharmaceutical examples and the rarity of unintended immunosuppression, routine immune function testing of all new pharmaceuticals is not considered justified. Resources currently being employed in this manner in an attempt to detect a seemingly rare phenomenon would appear to be better applied to the development of reliable predictive assays for drug hypersensitivity, which is known to cause significant patient morbidity. Any moves towards a globally harmonised guideline that recommends the use of concern-based IFTs, need ideally to be accompanied by the establishment of appropriate historical control reference intervals and interpretation criteria to support a reliable weight-of-evidence approach to data evaluation. |
15546687 | Legal constraints in EU product labelling to mitigate the environmental risk of veterinary medicines at use. | This paper summarises what possibilities and obligations are created by the EU Directive 2001/82/EC on the registration of veterinary medicines to mitigate the environmental impact of the use of a veterinary medicinal product. More specifically, an identified environmental risk may be mitigated to an acceptable level by special precautions in the information that accompanies the product in labelling and packaging. These precautions can address the fate of contaminated slurry and treated animals, but are only acceptable under the EU Directive if their effect can be demonstrated using the risk assessment methodology. Next, all possible keepers of the animals or the manure, including third parties, should be addressed, either in the precaution, or in the national regulation that should enforce the precautions. A number of examples illustrate that some precautions used are not quantifiable in the risk methodology, and that others are legally inadequate. To render risk mitigation measures effective, hence suitable for labelling and packaging, it is imperative that the risk assessment methodology is further developed and applied adequately, and that the legality of precautions is established in national regulation, harmonised between Member States. |
15546686 | Ciprofloxacin at low levels disrupts colonization resistance of human fecal microflora growing in chemostats. | We studied the in vitro effects of a range of ciprofloxacin (CI) concentrations on the human intestinal flora's colonization resistance (CR) to Salmonella kedougou NCTC 12173. Four steady state microbial communities were established in chemostats using inocula from a single pool of human feces. Three chemostats were exposed to CI (0.1, 0.43 and 5 microg/mL, respectively); one served as a no-drug control. The CR of each community was tested by three successive daily challenges of 10(8) S. kedougou, each delivered in a 1 mL bolus. There was no colonization of the no-drug chemostat. Likewise, after exposure to only 0.1 microg/mL CI there was no loss of CR and S. kedougou did not colonize. Conversely, both the 0.43 and the 5 microg/mL-exposed floras suffered a loss of CR and these chemostats were colonized. S. kedougou overgrew faster and reached higher counts in the presence of 0.43 than it did in the presence of 5 microg/mL. One possible explanation is that CI had a dose-dependent effect on both the challenge strain and CR. Thus, at higher levels, even though CR was disrupted by CI, so too was the growth of the challenge strain. Since exposure to CI elicited a dose-dependent reduction in Escherichia coli counts [Reg. Pharmacol. Toxicol. 33 (2001) 276] our new data suggest that E. coli may contribute to the CR against salmonella. We further conclude that, even at fecal levels below those reached during therapy, CI may impact the human gut flora sufficiently to facilitate colonization by S. kedougou. |
15546685 | Improving protection for research subjects in France: analysis of regional ethics committees. | The aim of this study was to analyze the functioning of the French committees for the protection of people in biomedical research (CCPPRB). An independent evaluator visited all of the committees and analyzed their functioning by assessing the annual activity reports, filling data grids and observing the committees in session. We observed that the representation of a range of professions, which is required by law, was not always respected. This was partly because the administrative authority had accumulated delays in nominating members. Another explanation could be the absence of remuneration for the work and attendance of the members. There was considerable inter-committee variation concerning the way documents were distributed to the members during sessions and the extent of investigator and promoter involvement. Finally, large differences in the number of dossiers handled between committees led us to investigate the fairness of dossier distribution, as the number of dossiers dealt with has consequences, particularly for the finality of the opinions of the committees. This evaluation of the committees playing a crucial role in protecting participants in research trials provides new information that could be helpful for improving the way in which these committees function in the context of the European Directive. |
15546684 | Toxicological resources for cumulative risk: an example with hazardous air pollutants. | Cumulative risk assessment, concerned with the multiple health effects of chemical mixtures, challenges the utility of existing single-chemical regulatory references. We compare example cumulative risk assessments for 40 HAPs; one based on single-effect toxicological data from EPA, and another based on a multiple-effect toxicological database we developed. For the 40-chemical HAP subset, the multiple effect database contains information on approximately seven effects per chemical and contains a total of 290 toxicological values. Seven health effects are represented in the IRIS data. Seventeen health effects are represented in the multiple-effect data. Respiratory and neurological effects ranked first and second in both cumulative analyses, regardless of the source data. In addition to respiratory and neurological effects, gastro-intestinal/hepatic, renal/kidney, and immunologic effects were identified as effects of concern on the basis of the multiple effect data. Immunologic effects are not found in the 40-chemical IRIS dataset. Cumulative risk assessment has the potential to expand our understanding of the public health impacts of environmental exposures. Advancements in toxicological resources will improve cumulative risk assessment. Cumulative risk assessment will reduce risks to the extent that it can be integrated into prevention strategies to track and protect the public's health. |
15546683 | The stability of historical control data for common neoplasms in laboratory rats and the implications for carcinogenic risk assessment. | Time-related changes in the incidences of spontaneous neoplasms in skin (fibroma and keratoacanthoma), thyroid (C-cell and follicular cell adenomas/carcinomas), uterus (stromal polyp), testes (Leydig cell tumor) and hemolymphoreticular system (mesenteric lymph node hemangioma and malignant granular lymphocytic leukemia) were assessed statistically in Wistar, Sprague-Dawley and F344 rats employed by the BASF, Germany and major European contract research organizations over the last 20 years. Negative trends (5 out of 80 cases) were observed for skin fibromas in F344 males, for follicular cell adenomas in Han Wistar females and in Sprague-Dawley males and females, and for follicular cell carcinomas in Sprague-Dawley males. Positive trends (8 out of 80 cases) were observed for skin keratoacanthomas in Han Wistar males, for C-cell adenomas in BASF Wistar males and females, for stromal polyps in Han Wistar and Sprague-Dawley females, and for mesenteric lymph node hemangiomas in Han Wistar and Sprague-Dawley males and in BASF Wistar females. In 67 out of 80 cases there were no statistically significant trends. Tumor drift was not common but occurred far more often in outbred rat strains (Wistar and Sprague-Dawley) than in the inbred rat strain (F344). This observation suggests that tumor predisposition is genetically determined, that tumor drift is primarily caused by genetic drift and that non-genotoxic carcinogens operate by facilitating the expression of tumor predisposition in target cells. |
15546682 | Toxicity evaluation of a beta-galactosidase preparation produced by Penicillium multicolor. | Tilactase is a beta-galactosidase enzyme preparation having lactase activity produced from the fungus Penicillium multicolor. The safety of tilactase was investigated in rats, dogs, and rabbits. Adult and juvenile rats administered 0, 500, 1000, or 4000 mg/kg bw/day of the enzyme preparation by gavage for 35 days, and dogs administered 0, 200, 500, or 1000 mg/kg bw/day in capsules for 30 days, exhibited no significant dose-related changes in body weights, feed consumption, organ weights, urinalysis, hematological profiles, clinical chemistry, or histopathological profiles. Rats receiving the same doses for 6 months also exhibited no dose-related effects, except for a small increase in the weight of the large intestine, an effect considered to be a physiological reaction to passage of a large amount of a non-absorbable substance. The no-observable-adverse-effect level (NOAEL) was 4000 mg/kg bw/day for rats and 1000 mg/kg bw/day for dogs. In three separate studies to examine reproductive and developmental toxicity, rats received 0, 250, 1000 or 4000 mg/kg bw/day by gavage up to the 7th day of pregnancy, during days 7-17 of pregnancy, and from day 17 of pregnancy to 21 days after delivery. There were no treatment-related effects on the dams, gestation period, numbers of implantations, parturition rates, sex ratios, or survival of offspring in any of the studies. No treatment-related external, internal, or skeletal abnormalities were observed in fetuses from any of the three studies. The NOAEL was 4000 mg/kg bw/day. In addition to the three rat studies, rabbits received 0, 250, 500, or 1000 mg/kg bw/day by gavage from the 6th to 18th day of pregnancy. No treatment-related changes were observed in the dams, or fertility indices; nor were there any treatment-related fetal abnormalities. The NOAEL was 1000 mg/kg bw/day. When viable P. multicolor spores were injected into the tail veins of mice, no deaths occured, no fungal cells were observed in various organs, and histopathology showed only focal necrosis in the liver of some of the animals, including the controls. Similar effects were observed when spores were administered to mice in a single dose by gavage. The particular strain of P. multicolor used to prepare tilactase is not pathogenic. |
15546681 | An intervention analysis for the reduction of exposure to methylmercury from the consumption of seafood by women of child-bearing age. | A previously developed exposure model was used [Risk Anal. 22 (2002) 689] to assess the effectiveness of various advisory scenarios on minimizing mercury (Hg) blood levels via the consumption of commercial seafood, both finfish and shellfish. This exposure model was developed to predict levels of Hg in blood in women of child-bearing age in the US based on the frequency of seafood consumption, the amount of seafood consumed per serving, and the types of seafood consumed. Steady-state relationships that employed descriptive statistics to account for toxicokinetic variation were used to predict levels of Hg in blood. The model incorporates an uncertainty dimension that is intended to represent the range of plausible interpretations of the data. The predictability of the model was confirmed via the use of National Health and Nutrition Examination Survey (NHANES) blood Hg data. In the present analysis, the model was used to predict the impact of limitations in the amount or types of seafood consumed on blood Hg levels. Specifically, simulations for various advisory scenarios were developed on the basis of limitations on total consumption of seafood, elimination of the consumption of certain species altogether, and/or a combination of both. In the baseline model, the median (uncertainty) estimates for the 50th, 95th, and 99th per capita population percentiles were 1.25, 8.2, and 16.1 ppb blood Hg, respectively. After restriction of seafood consumption to no more than 12 oz/week, the median (uncertainty) estimates for the 50th, 95th, and 99th per capita population percentiles were 1.22, 6.8, and 10.6 ppb blood Hg, respectively. Elimination of MeHg species, with average concentrations above 0.6 ppm, resulted in very modest decrements in Hg blood levels, in comparison to either the baseline or the reduced consumption scenarios. These results suggest that strategies to reduce MeHg exposure by reducing the amount of fish consumed (e.g., 12 oz/week) are more effective at eliminating the high end of the exposure distribution than are strategies intended to change the types of fish consumed. |
15546680 | Magnitude and mechanistic determinants of the interspecies toxicokinetic uncertainty factor for organic chemicals. | The interspecies uncertainty factor, UF(AH), of 10 was recently subdivided into two components to account separately for interspecies differences in toxicokinetics and toxicodynamics (UF(AH-TK)=3.16, UF(AH-TD)=3.16). Even though the UF(AH) in its composite or dissociated form is used during the health risk assessment of systemic toxicants, there is no convincing scientific basis to justify the use of the same UF for all chemicals. In this study, we use equations that describe the toxicokinetics of chemicals at steady-state to characterize the magnitude and mechanistic determinants of UF(AH-TK) for several volatile organic chemicals (VOCs). Further, algorithms have been developed to determine the magnitude of the components of UF(AH-TK), namely the UF(AH-TK-ABS) (accounting for interspecies differences in dose absorbed during identical inhalation exposure conditions), UF(AH-TK-MET) (referring to the factor by which the blood concentration of unchanged parent chemical differs from one species to another, due to metabolic clearance, when both species receive identical doses) and UF(AH-TK-DIS) (reflecting the magnitude of difference in chemical concentrations distributed in target tissues of two species when the arterial blood concentration in both species is identical). The results show that the body weight, the rate of alveolar ventilation, the fraction of cardiac output flowing to the liver, partition coefficients (blood:air and tissue:blood), and the hepatic extraction ratio are the only parameters that play a critical role in the extrapolation of tissue and blood concentrations across species. Further, the magnitude of the UF(AH-TK-ABS) (means+/-SD, 0.19+/-0.04), UF(AH-TK-MET) (means+/-SD, 0.24+/-0.05) and UF(AH-TK-DIS) (mean range: 1.76-0.93) obtained in this study for several VOCs compares well with that obtained previously using physiologically based toxicokinetic models. |
15546679 | Statistical methodology to evaluate food exposure to a contaminant and influence of sanitary limits: application to Ochratoxin A. | This paper presents some statistical methodologies to evaluate the food exposure to a contaminant and quantify the outcome of a new maximum limit on a food item. Our application deals with Ochratoxin A (OTA). We focus on the quantitative evaluation of the distribution of exposure based on both consumption data and contamination data. One specific aspect of contamination data is left censorship due to the limits of detection. Three calculation procedures are proposed: [P1] a deterministic method using means of contamination; [P2] a probabilistic method using a parametric adjustment of the distributions of contamination taking into account the left censorship; and [P3] a non-parametric method which consists in randomly selecting the consumption data and the contamination values. Our main result shows that a non-parametric probabilistic approach is well adapted for the purpose of exposure assessment, when large samples are available. In the application to OTA, the probability to exceed a safe level is high, particularly for children. Simulations show that the impact of the existing standards on cereals and the currently proposed standards on wine generally do not significantly reduce the risk to be overexposed to OTA. |
15546678 | A risk-based methodology for deriving quality standards for organic contaminants in sewage sludge for use in agriculture--Conceptual Framework. | This paper describes a systematic methodology (Conceptual Framework) to derive quality standards for organic (anthropogenic) contaminants in sewage sludge added to agricultural land, in the context of revision of EU Sludge Directive 86/278/EEC and the broader Soil Thematic Strategy. The overall objective is to ensure, based on a risk assessment approach, a sustainable use of sludge over a long time horizon. ILSI-Europe's Conceptual Framework is in essence consistent with the EU Technical Guidance Document (TGD) for Environmental Risk Assessment of Chemicals in the soil compartment, or US-EPA's Sewage Sludge Use and Disposal Regulations, Part 503 Standards. A 'checklist' of different exposure pathways and transfer processes for organic contaminants needs to be considered, and the most sensitive relevant toxicological endpoint and its PNEC need to be identified. The additional complexity specific to deriving Sludge Quality Standards (SQS) is that the toxicity results may need-e.g., for (indirect) human toxicity-to be related back to maximum acceptable soil exposure levels (PEC(soil)). In turn, the latter need to be back-calculated to the maximum acceptable levels in sewage sludge (PEC(sludge)) at the time of application. Finally, for a sustainable sludge use, the exposure from repeated addition and potential chemical build-up over time (e.g., 100 years) needs to be assessed. The SQS may therefore vary with the (local) sludge application regime, and/or sludge pretreatment processes. |
15546677 | Evaluation of a surrogate antibody for preclinical safety testing of an anti-CD11a monoclonal antibody. | Surrogate antibodies are a potential solution to the limited safety testing possible with humanized monoclonal antibodies with restricted species cross-reactivity. However, there are currently no defined criteria by which a potential surrogate antibody should be judged prior to its use in determining safety issues for the clinical agent. We propose that, potential surrogates should undergo rigorous evaluation to assess pharmacological and toxicological activities in comparison to the clinical agent. The current studies evaluated a chimeric mouse/rat anti-mouse CD11a monoclonal antibody (muM17) as a potential surrogate for efalizumab, a humanized anti-CD11a antibody in development for psoriasis. CD11a is a subunit of lymphocyte function associated antigen-1, an integrin involved in cell-cell interactions important to immune responses and inflammation. In vitro pharmacology studies included binding affinity to whole mouse blood and inhibitory activity of muM17 in a mixed lymphocyte response assay. In vivo pharmacology was examined using a delayed type hypersensitivity assay in female CD-1 mice. The toxicology evaluation included a murine tissue cross-reactivity study and in vivo multiple dose studies in female CD-1 mice which were administered muM17 (0.1-30 mg/kg) via subcutaneous injections once a week for 4 weeks. Clinical observations, body weight, clinical pathology, T cell CD11a expression, immunogenicity, toxicokinetics, and lymphoid organ histopathology were evaluated. Finally, since reproductive safety testing would be an important application of the proposed surrogate antibody, a pilot study in pregnant mice was conducted that demonstrated proportional transfer of muM17 into the fetus. These studies demonstrated that muM17 has pharmacological and toxicological activities similar to efalizumab. The selection of dose and regimen for GLP (Good Laboratory Practice) toxicology studies and extrapolation to clinical dose levels was based on pharmacodynamic activity (CD11a downmodulation on T cells). |
15546676 | Synchronous evaluation of toxico- and pharmaco-dynamics of yttrium-90 by a novel autoradiographic procedure. | A synchronous evaluation was performed, using a quick in vivo [2-(14)C]thymidine labeling method, of the toxico- and pharmaco-dynamics of a given dose of yttrium-90 (90Y) at a given time after injection to nude BALB/c mice loaded with 10(7) HuO9 cells. Quantitative data were 14C-microautoradiographs of the liver lobule, intestinal crypts, epiphysial growth plate, secondary ossification center containing pluripotent stem cells, perifollicular zone containing unipotent stem cells in the spleen, and plasmacytoma cells in the osteogenic sarcoma in each mouse following a 10-min labeling with 14C at 0.5, 6, and 24 h after i.v. injection of 90Y. Results show that the cell proliferation rate of the stem cells in respective tissues was markedly suppressed, dependent on time after dosing and the dose of 90Y; 3.7, 37, 370, 3700, and 37,000 kBq per mouse (25 g). In addition to the above, the sensitivity of the proliferation rate was dependent on amitosis or mitosis and the AUC value of 90Y-concentration at specific locations of the cells in the mouse body. The most sensitive cells were the plasmacytoma cells, followed by the pluripotent and unipotent stem cells, the intestinal crypts, epiphysial growth plate, and liver cells. |
15546675 | Estimating the safe starting dose in phase I clinical trials and no observed effect level based on QSAR modeling of the human maximum recommended daily dose. | Estimating the maximum recommended starting dose (MRSD) of a pharmaceutical for phase I human clinical trials and the no observed effect level (NOEL) for non-pharmaceuticals is currently based exclusively on an extrapolation of the results of animal toxicity studies. This process is inexact and requires the results of toxicity studies in multiple species (rat, dog, and monkey) to identify the no observed adverse effect level (NOAEL) and most sensitive test species. Multiple uncertainty (safety) factors are also necessary to compensate for incompatibility and uncertainty underlying the extrapolation of animal toxicity to humans. The maximum recommended daily dose for pharmaceuticals (MRDD) is empirically derived from human clinical trials. The MRDD is an estimated upper dose limit beyond which a drug's efficacy is not increased and/or undesirable adverse effects begin to outweigh beneficial effects. The MRDD is essentially equivalent to the NOAEL in humans, a dose beyond which adverse (toxicological) or undesirable pharmacological effects are observed. The NOAEL in test animals is currently used to estimate the safe starting dose in human clinical trials. MDL QSAR predictive modeling of the human MRDD may provide a better, simpler and more relevant estimation of the MRSD for pharmaceuticals and the toxic dose threshold of chemicals in humans than current animal extrapolation based risk assessment models and may be a useful addition to current methods. A database of the MRDD for over 1300 pharmaceuticals was compiled and modeled using MDL QSAR software and E-state and connectivity topological descriptors. MDL QSAR MRDD models were found to have good predictive performance with 74-78% of predicted MRDD values for 120 internal and 160 external validation compounds falling within a range of +/-10-fold the actual MRDD value. The predicted MRDD can be used to estimate the MRSD for pharmaceuticals in phase I clinical trials with the addition of a 10-fold safety factor. For non-pharmaceutical chemicals any compound-related effect can be considered an undesirable and adverse toxicological effect and the predicted MRDD can be used to estimate the NOEL with the addition of an appropriate safety factor. |
15546674 | Post-translocational folding of secretory proteins in Gram-positive bacteria. | The transport of proteins from their site of synthesis in the cytoplasm to their functional location is an essential characteristic of all living cells. In Gram-positive bacteria the majority of proteins that are translocated across the cytoplasmic membrane are delivered to the membrane-cell wall interface in an essentially unfolded form. They must then be folded into their native configuration in an environment that is dominated by a high density of immobilised negative charge-in essence an ion exchange resin. It is essential to the viability of the cell that these proteins do not block the translocation machinery in the membrane, form illegitimate interactions with the cell wall or, through intermolecular interactions, form insoluble aggregates. Native Gram-positive proteins therefore have intrinsic folding characteristics that facilitate their rapid folding, and this is assisted by a variety of folding factors, including enzymes, peptides and metal ions. Despite these intrinsic and extrinsic factors, secretory proteins do misfold, particularly if the cell is subjected to certain types of stress. Consequently, Gram-positive bacteria such as Bacillus subtilis encode membrane- and cell wall-associated proteases that act as a quality control machine, clearing misfolded or otherwise aberrant proteins from the translocase and the cell wall. |
15546673 | Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. | Bacillus subtilis is a rod-shaped, Gram-positive soil bacterium that secretes numerous enzymes to degrade a variety of substrates, enabling the bacterium to survive in a continuously changing environment. These enzymes are produced commercially and this production represents about 60% of the industrial-enzyme market. Unfortunately, the secretion of heterologous proteins, originating from Gram-negative bacteria or from eukaryotes, is often severely hampered. Several bottlenecks in the B. subtilis secretion pathway, such as poor targeting to the translocase, degradation of the secretory protein, and incorrect folding, have been revealed. Nevertheless, research into the mechanisms and control of the secretion pathways will lead to improved Bacillus protein secretion systems and broaden the applications as industrial production host. This review focuses on studies that aimed at optimizing B. subtilis as cell factory for commercially interesting heterologous proteins. |
15546672 | Type I signal peptidases of Gram-positive bacteria. | Proteins that are exported from the cytoplasm to the periplasm and outer membrane of Gram-negative bacteria, or the cell wall and growth medium of Gram-positive bacteria, are generally synthesized as precursors with a cleavable signal peptide. During or shortly after pre-protein translocation across the cytoplasmic membrane, the signal peptide is removed by signal peptidases. Importantly, pre-protein processing by signal peptidases is essential for bacterial growth and viability. This review is focused on the signal peptidases of Gram-positive bacteria, Bacillus and Streptomyces species in particular. Evolutionary concepts, current knowledge of the catalytic mechanism, substrate specificity requirements and structural aspects are addressed. As major insights in signal peptidase function and structure have been obtained from studies on the signal peptidase LepB of Escherichia coli, similarities and differences between this enzyme and known Gram-positive signal peptidases are highlighted. Notably, while the incentive for previous research on Gram-positive signal peptidases was largely based on their role in the biotechnologically important process of protein secretion, present-day interest in these essential enzymes is primarily derived from the idea that they may serve as targets for novel anti-microbials. |
15546671 | Protein sorting to the cell wall envelope of Gram-positive bacteria. | The covalent anchoring of surface proteins to the cell wall envelope of Gram-positive bacteria occurs by a universal mechanism requiring sortases, extracellular transpeptidases that are positioned in the plasma membrane. Surface protein precursors are first initiated into the secretory pathway of Gram-positive bacteria via N-terminal signal peptides. C-terminal sorting signals of surface proteins, bearing an LPXTG motif or other recognition sequences, provide for sortase-mediated cleavage and acyl enzyme formation, a thioester linkage between the active site cysteine residue of sortase and the C-terminal carboxyl group of cleaved surface proteins. During cell wall anchoring, sortase acyl enzymes are resolved by the nucleophilic attack of peptidoglycan substrates, resulting in amide bond formation between the C-terminal end of surface proteins and peptidoglycan cross-bridges within the bacterial cell wall envelope. The genomes of Gram-positive bacteria encode multiple sortase genes. Recent evidence suggests that sortase enzymes catalyze protein anchoring reactions of multiple different substrate classes with different sorting signal motif sequences, protein linkage to unique cell wall anchor structures as well as protein polymerization leading to the formation of pili on the surface of Gram-positive bacteria. |
15546668 | Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems. | The translocation of DNA across biological membranes is an essential process for many living organisms. In bacteria, type IV secretion systems (T4SS) are used to deliver DNA as well as protein substrates from donor to target cells. The T4SS are structurally complex machines assembled from a dozen or more membrane proteins in response to environmental signals. In Gram-negative bacteria, the conjugation machines are composed of a cell envelope-spanning secretion channel and an extracellular pilus. These dynamic structures (i) direct formation of stable contacts-the mating junction-between donor and recipient cell membranes, (ii) transmit single-stranded DNA as a nucleoprotein particle, as well as protein substrates, across donor and recipient cell membranes, and (iii) mediate disassembly of the mating junction following substrate transfer. This review summarizes recent progress in our understanding of the mechanistic details of DNA trafficking with a focus on the paradigmatic Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation systems. |
15546670 | Fiber assembly by the chaperone-usher pathway. | Bacterial pathogens utilize the chaperone-usher pathway to assemble extracellular multi-subunit fibers essential for virulence. The periplasmic chaperone facilitates the initial folding of fiber subunits but then traps them in activated folding transition states. Chaperone dissociation releases the folding energy that drives subunit incorporation into the fiber, which grows through a pore formed by the outer-membrane usher. |
15546669 | Protein secretion through autotransporter and two-partner pathways. | Two distinct protein secretion pathways, the autotransporter (AT) and the two-partner secretion (TPS) pathways are characterized by their apparent simplicity. Both are devoted to the translocation across the outer membrane of mostly large proteins or protein domains. As implied by their name, AT proteins contain their own transporter domain, covalently attached to the C-terminal extremity of the secreted passenger domain, while TPS systems are composed of two separate proteins, with TpsA being the secreted protein and TpsB its specific transporter. In both pathways, the secreted proteins are exported in a Sec-dependent manner across the inner membrane, after which they cross the outer membrane with the help of their cognate transporters. The AT translocator domains and the TpsB proteins constitute distinct families of protein-translocating, outer membrane porins of Gram-negative bacteria. Both types of transporters insert into the outer membrane as beta-barrel proteins possibly forming oligomeric pores in the case of AT and serve as conduits for their cognate secreted proteins or domains across the outer membrane. Translocation appears to be folding-sensitive in both pathways, indicating that AT passenger domains and TpsA proteins cross the periplasm and the outer membrane in non-native conformations and fold progressively at the cell surface. A major difference between AT and TPS pathways arises from the manner by which specificity is established between the secreted protein and its transporter. In AT, the covalent link between the passenger and the translocator domains ensures the translocation of the former without the need for a specific molecular recognition between the two modules. In contrast, the TPS pathway has solved the question of specific recognition between the TpsA proteins and their transporters by the addition to the TpsA proteins of an N-proximal module, the conserved TPS domain, which represents a hallmark of the TPS pathway. |
15546667 | Type III flagellar protein export and flagellar assembly. | Bacterial flagella, unlike eukaryotic flagella, are largely external to the cell and therefore many of their subunits have to be exported. Export is ATP-driven. In Salmonella, the bacterium on which this chapter largely focuses, the apparatus responsible for flagellar protein export consists of six membrane components, three soluble components and several substrate-specific chaperones. Other flagellated eubacteria have similar systems. The membrane components of the export apparatus are housed within the flagellar basal body and deliver their substrates into a channel or lumen in the nascent structure from which point they diffuse to the far end and assemble. Both on the basis of sequence similarities of several components and structural similarities, the flagellar protein export systems clearly belong to the type III superfamily, whose other members are responsible for secretion of virulence factors by many species of pathogenic bacteria. |
15546666 | Type III protein secretion mechanism in mammalian and plant pathogens. | The type III protein secretion system (TTSS) is a complex organelle in the envelope of many Gram-negative bacteria; it delivers potentially hundreds of structurally diverse bacterial virulence proteins into plant and animal cells to modulate host cellular functions. Recent studies have revealed several basic features of this secretion system, including assembly of needle/pilus-like secretion structures, formation of putative translocation pores in the host membrane, recognition of N-terminal/5' mRNA-based secretion signals, and requirement of small chaperone proteins for optimal delivery and/or expression of effector proteins. Although most of our knowledge about the TTSS is derived from studies of mammalian pathogenic bacteria, similar and unique features are learned from studies of plant pathogenic bacteria. Here, we summarize the most salient aspects of the TTSS, with special emphasis on recent findings. |
15546665 | The underlying mechanisms of type II protein secretion. | The cell envelope of Gram-negative bacteria is composed of two membranes, which are separated by the peptidoglycan-containing periplasm. Whereas the envelope forms an essential barrier against harmful substances, it is nevertheless a compartment of intense traffic for large proteins such as enzymes and toxins. Numerous studies dealing with the molecular mechanism of protein secretion have revealed that Gram-negative bacteria evolved different strategies to achieve this process. Among them, the type II secretion mechanism is part of a two-step process. Exoproteins following this pathway are synthesized as signal peptide-containing precursors. After cleavage of the signal peptide, the mature exoproteins are released into the periplasm, where they fold. The type II machinery, also known as the secreton, is responsible for the translocation of the periplasmic intermediates across the OM. The type II system is broadly conserved in Gram-negative bacteria and involves a set of 12-16 different proteins named GspC-M, GspAB, GspN, GspO, and GspS. The type II secretion system is highly reminiscent of the type IV piliation assembly system. Based on findings about the subcellular localisation of the Gsp components, protein-protein interactions between Gsps and their multimerisation status, structural data and electron microscopy observation, it could be proposed a working model that strikingly runs both systems in parallel. |
15546664 | Type I secretion in gram-negative bacteria. | In gram-negative bacteria, type I secretion is carried out by a translocator made up of three proteins that span the cell envelope. One of these proteins is a specific outer membrane protein (OMP) and the other two are cytoplasmic membrane proteins: an ATP-binding cassette (ABC) and the so-called membrane fusion or adaptor protein (MFP). Type I secretion is sec-independent and bypasses the periplasm. This widespread pathway allows the secretion of proteins of diverse sizes and functions via a C-terminal uncleaved secretion signal. This C-terminal secretion signal specifically recognizes the ABC protein, triggering the assembly of the functional trans-envelope complex. This report will mainly deal will recent data concerning the structure and assembly of the secretion complex as well as the effects and role of substrate folding on secretion by this pathway. |
15546663 | Tat-dependent protein targeting in prokaryotes and chloroplasts. | The twin-arginine translocation (Tat) system operates in the chloroplast thylakoid and the plasma membranes of a wide range of bacteria. It recognizes substrates bearing cleavable signal peptides in which a twin-arginine motif almost invariably plays a key role in recognition by the translocation machinery. These signal peptides are surprisingly similar to those used to specify transport by Sec-type systems, but the Tat pathway differs in fundamental respects from Sec-type and other protein translocases. Its key attribute is its ability to translocate large, fully folded (even oligomeric) proteins across tightly sealed membranes. To date, three key tat genes have been characterised and the first details of the Tat system are beginning to emerge. In this article we review the salient features of Tat systems, with an emphasis on the targeting signals involved, the substrate specificities of Tat systems, our current knowledge of Tat complex structures and the known mechanistic features. Although the article is focused primarily on bacterial systems, we incorporate relevant aspects of plant thylakoid Tat work and we discuss how the plant and bacterial systems may differ in some respects. |
15546662 | Quality control in the bacterial periplasm. | Studies of the mechanisms that Gram-negative bacteria use to sense and respond to stress have led to a greater understanding of protein folding in both cytoplasmic and extracytoplasmic locations. In response to stressful conditions, bacteria induce a variety of stress response systems, examples of which are the sigma(E) and Cpx systems in Escherichia coli. Induction of these stress response systems results in upregulation of several gene targets that have been shown to be important for protein folding under normal conditions. Here we review the identification of stress response systems and their corresponding gene targets in E. coli. In addition, we discuss the apparent redundancy of the folding factors in the periplasm, and we consider the potential importance of the functional overlap that exists. |
15546661 | Catalysis of disulfide bond formation and isomerization in the Escherichia coli periplasm. | Disulfide bond formation is a catalyzed process in vivo. In prokaryotes, the oxidation of cysteine pairs is achieved by the transfer of disulfides from the highly oxidizing DsbA/DsbB catalytic machinery to substrate proteins. The oxidizing power utilized by this system comes from the membrane-embedded electron transport system, which utilizes molecular oxygen as a final oxidant. Proofreading of disulfide bond formation is performed by the DsbC/DsbD system, which has the ability to rearrange non-native disulfides to their native configuration. These disulfide isomerization reactions are sustained by a constant supply of reducing power provided by the cytoplasmic thioredoxin system, utilizing NADPH as the ultimate electron source. |
15546660 | The role of lipids in membrane insertion and translocation of bacterial proteins. | Phospholipids are essential building blocks of membranes and maintain the membrane permeability barrier of cells and organelles. They provide not only the bilayer matrix in which the functional membrane proteins reside, but they also can play direct roles in many essential cellular processes. In this review, we give an overview of the lipid involvement in protein translocation across and insertion into the Escherichia coli inner membrane. We describe the key and general roles that lipids play in these processes in conjunction with the protein components involved. We focus on the Sec-mediated insertion of leader peptidase. We describe as well the more direct roles that lipids play in insertion of the small coat proteins Pf3 and M13. Finally, we focus on the role of lipids in membrane assembly of oligomeric membrane proteins, using the potassium channel KcsA as model protein. In all cases, the anionic lipids and lipids with small headgroups play important roles in either determining the efficiency of the insertion and assembly process or contributing to the directionality of the insertion process. |
15546659 | The protein-conducting channel SecYEG. | In bacteria, the translocase mediates the translocation of proteins into or across the cytosolic membrane. It consists of a membrane embedded protein-conducting channel and a peripherally associated motor domain, the ATPase SecA. The channel is formed by SecYEG, a multimeric protein complex that assembles into oligomeric forms. The structure and subunit composition of this protein-conducting channel is evolutionary conserved and a similar system is found in the endoplasmic reticulum of eukaryotes and the cytoplasmic membrane of archaea. The ribosome and other membrane proteins can associate with the protein-conducting channel complex and affect its activity or functionality. |
15546658 | Structure and function of SecA, the preprotein translocase nanomotor. | Most secretory proteins that are destined for the periplasm or the outer membrane are exported through the bacterial plasma membrane by the Sec translocase. Translocase is a complex nanomachine that moves processively along its aminoacyl polymeric substrates effectively pumping them to the periplasmic space. The salient features of this process are: (a) a membrane-embedded "clamp" formed by the trimeric SecYEG protein, (b) a "motor" provided by the dimeric SecA ATPase, (c) regulatory subunits that optimize catalysis and (d) both chemical and electrochemical metabolic energy. Significant recent strides have allowed structural, biochemical and biophysical dissection of the export reaction. A model incorporating stepwise strokes of the translocase nanomachine at work is discussed. |
15546657 | Membrane integration of E. coli model membrane proteins. | The molecular events of membrane translocation and insertion have been investigated using a number of different model proteins. Each of these proteins has specific features that allow interaction with the membrane components which ensure that the proteins reach their specific local destination and final conformation. This review will give an overview on the best-characterized proteins studied in the bacterial system and emphasize the distinct aspects of the pathways. |
15546656 | Sec-translocase mediated membrane protein biogenesis. | Alpha-helical transmembrane proteins in bacteria are localized within the plasma membrane. The membrane assembly of these proteins requires protein devices for insertion into the lipid bilayer. In E. coli, membrane proteins require the SRP pathway components Ffh, 4.5S RNA and FtsY for membrane targeting and the SecYEGDF translocase and, in some cases, SecA, for translocation of hydrophilic domains. In addition, YidC, a recently discovered membrane protein, mediates the membrane integration and folding of hydrophobic domains of membrane proteins. In this review, we will describe the current status of the protein targeting and membrane integration pathways. |
15546655 | SRP-mediated protein targeting: structure and function revisited. | The signal recognition particle (SRP) and its membrane-bound receptor (SR) deliver membrane proteins and secretory proteins to the translocation channel in the plasma membrane (or the endoplasmic reticulum). The general outline of the SRP pathway is conserved in all three kingdoms of life. During the past decade, structure determination together with functional studies has brought our understanding of the SRP-mediated protein transport to an almost molecular level. An impressive amount of new information especially on the prokaryotic SRP is integrated into the current picture of the SRP pathway. |
15546654 | Translocation of bacterial proteins--an overview. | Recent progress in the understanding of the nature of the extraordinary variety of protein translocation systems, mainly in Gram negative bacteria, is reviewed. This takes us from the insertion of proteins into the inner membrane via the sophisticated Sec apparatus, the lethal injection of Type III proteins into host cells and on to the beautiful machine that assembles the flagellum. Attempts are made to establish some order, some common principles that might explain the variety and the complexity of some systems. The fundamentals considered are the nature of different transport signals, the nature of translocons (a wide variety of inner membrane types, outer membrane translocons are more conserved), the process of docking to translocons, the role of chaperones and the folding of transported proteins, the energetics of translocation, and prospects for future advances. |
15546651 | Acupuncture: a promising treatment for depression during pregnancy. | Few medically acceptable treatments for depression during pregnancy are available. The aim of this randomized controlled pilot study was to determine whether acupuncture holds promise as a treatment for depression during pregnancy. Sixty-one pregnant women with major depressive disorder and a 17-item Hamilton Rating Scale for Depression (HRSD17) score >or=14 were randomly assigned to one of three treatments, delivered over 8 weeks: an active acupuncture (SPEC, N=20), an active control acupuncture (NSPEC, N=21), and massage (MSSG, N=20). Acupuncture treatments were standardized, but individually tailored, and were provided in a double-blind fashion. Responders to acute phase treatment (HRSD17 score<14 and >or=50% reduction from baseline) continued the treatment they were initially randomized to until 10 weeks postpartum. Response rates at the end of the acute phase were statistically significantly higher for SPEC (69%) than for MSSG (32%), with an intermediate NSPEC response rate (47%). The SPEC group also exhibited a significantly higher average rate of reduction in BDI scores from baseline to the end of the first month of treatment than the MSSG group. Responders to the acute phase of all treatments combined had significantly lower depression scores at 10 weeks postpartum than nonresponders. Generalizability is limited by the small sample and its relative homogeneity. Acupuncture holds promise for the treatment of depression during pregnancy. |
15546650 | Prevalence of social phobia in a clinical sample of drug dependent patients. | Social phobia is among the most frequent psychiatric disorders and can be classified into two subtypes, nongeneralized and generalized. Whereas it significantly worsens the morbidity of comorbid substance abuse disorders, and it often is associated with reduced treatment responses, there is still lacking data on its prevalence in clinical populations of drug abusing patients. The study sample consisted of 75 inpatients and 75 outpatients meeting DSM-IV criteria for drug dependence. Symptoms of social phobia were assessed with the French-language version of the Liebowitz Social Anxiety Scale (LSAS). Prevalence rate were 20% for the generalized subtype and 42.6% for the nongeneralized subtype. Gender difference emerged in the severity of fear, women reporting significantly greater fear relating to performance situations than men. An important proportion of patients with substance dependence present a comorbid generalized or nongeneralized social phobia. Early recognition of social phobia and adequate interventions is warranted for these patients in order to improve their treatment response with regard to quality of life and relapse prevention. |
15546649 | Aberration of cholesterol level in first-onset bipolar I patients. | Cholesterol interacts with serotonin and it has been found to be associated with some clinical symptoms of mood disorders. There is a paucity of data on first-onset bipolar patients and from Asian population. The total cholesterol (TC) level was examined in 25 bipolar I patients with a single manic episode (BPSM) and 30 controls. The TC level was significantly lower in the BPSM than in the controls. There were negative correlations between the Young mania rating scale (YMRS) scores and the pretreatment TC level in BPSM. This study suggests that the TC level can be changed after treatment in bipolar manic patients, although more studies involving different ethnic groups will be needed. |
15546648 | Identifying suicidal ideation among older adults in a general practice setting. | Up to 70% of older people who commit suicide consult a general practitioner (GP) in the month prior to their death. The purpose of this study was to identify the clinical and demographic characteristics of older adults who are contemplating suicide and are in contact with a GP. We utilised a cross-sectional study to investigate the association between suicidal ideation and clinical/demographic variables of 504 consecutive patients aged 60 years or over, attending 1 of 54 randomly selected Western Australian GPs. Prior to their medical consultation, patients completed a self-report questionnaire, which included questions about suicidal ideation (Depressive Symptom Inventory-Suicidality Subscale, DSI-SS), demographic factors, lifestyle factors, physical health and mental health, including depression (Center for Epidemiologic Studies-Depression Scale, CES-D). Within our sample of older patients, 6.3% acknowledged current suicidal ideation. Multivariate analyses indicated that current suicidal ideation was strongly associated with being depressed at least occasionally during the previous week (OR=7.3, 95% CI=2.3-23.0), CES-D scores of 16 points or greater (OR=3.6, 95% CI=1.0-12.1), and a prior history of attempted suicide (OR=15.5, 95% CI=4.0-60.6). Our results and conclusions are limited to suicidal ideation, and may not apply to suicidal behaviour. Depressive symptomatology is strongly associated with suicidal ideation in later life. Strategies that enhance GPs' identification and treatment of affective illness in older patients should have the greatest impact on suicide rates within this age group. |
15546647 | Does type of first contact in depressive and bipolar disorders predict subsequent hospitalisation and risk of suicide? | Only a few studies have investigated how the type of first contact is associated with the risk of subsequent hospitalisation and the risk of committing suicide for patients with depressive or bipolar disorders. All outpatients (patients in psychiatric ambulatories and community psychiatry centres) and in-patients (patients admitted during daytime or overnight to a psychiatric hospital) with a diagnosis of depressive or bipolar disorder at first contact ever in a period from 1995 to 1999 in Denmark were identified from the Danish Psychiatric Central Research Register (DPCRR). The risk of subsequent hospitalisation and the risk of suicide were compared according to type of first contact. The risk of subsequent hospitalisation was significantly increased for patients who were admitted to inpatient facilities during first contact compared to patients with outpatient treatment as their first contact. Patients with depressive disorder who were admitted also had increased risk of committing suicide eventually. The diagnoses are clinician based. Patients referred to inpatient treatment have a poorer long-term prognosis than patients treated as outpatients. |
15546646 | Spatial processing of facial emotion in patients with unipolar depression: a longitudinal study. | In this study, a face-in-the-crowd task was applied to examine the spatial detection of facial emotion as a function of depression and comorbid anxiety in the course of a psychotherapeutic inpatient treatment. Patients with unipolar depression (n=22) and normal controls (n=22) were tested twice, about 7 weeks apart, on a face-in-the-crowd task using displays of schematic faces. Half the patients were suffering from a comorbid anxiety disorder. From test 1 to test 2, depressivity, frequency of negative thoughts, and worrying of patients improved significantly. At both sessions, depressed patients, irrespective of the presence of comorbid anxiety disorders, showed no performance differences in the detection of negative faces compared to controls. However, depressed patients with but not those without comorbid anxiety disorders were slower in responding to positive faces than controls. Both patient groups were slower in responding to the neutral faces' condition than controls. Our data indicate a spatial processing deficit for positive facial expression in depressives with a comorbid anxiety disorder. This impairment, which appears to persist during remission, might be due to deficits in effortful visual search processes. |
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