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991.
Formamidopyrimidine-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that excises oxidized purine bases, most notably the mutagenic 7-hydro-8-oxoguanine, from damaged DNA. In order to identify specific contacts between nucleobases of DNA and amino acids from the E. coli Fpg protein, photochemical cross-linking was employed using new reactive DNA duplexes containing 5-[4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]-2'-deoxyuridine dU* residues near the 7-hydro-8-oxoguanosine (oxoG) lesion. The Fpg protein was found to bind specifically and tightly to the modified DNA duplexes and to incise them. The nicking efficiency of the DNA duplex containing a dU* residue 5' to the oxoG was higher as compared to oxidized native DNA. The conditions for the photochemical cross-linking of the reactive DNA duplexes and the Fpg protein have been optimized to yield as high as 10% of the cross-linked product. Our results suggest that the Fpg protein forms contacts with two nucleosides, one 5' adjacent to oxoG and the other 5' adjacent to the cytidine residue pairing with oxoG in the other strand. The approaches developed may be applicable to pro- and eukaryotic homologues of the E. coli Fpg protein as well as to other repair enzymes.  相似文献   
992.
Neuropeptide Y (NPY), a sympathetic cotransmitter, acts via G protein-coupled receptors to stimulate constriction and vascular smooth muscle cell (VSMC) proliferation through interactions with its Y1 receptors. However, VSMC proliferation appears bimodal, with high- and low-affinity peaks differentially blocked by antagonists of both Y1 and Y5 receptors. Here, we sought to determine the signaling mechanisms of NPY-mediated bimodal mitogenesis. In rat aortic VSMCs, NPY's mitogenic effect at all concentrations was blocked by pertussis toxin and was associated with decreased forskolin-stimulated cAMP levels. NPY also increased intracellular calcium levels; in contrast to mitogenesis, this effect was dose dependent. The rise in intracellular Ca2+ depended on extracellular Ca2+ and was mediated via activation of Y1 receptors, but not Y5 receptors. Despite differences in calcium, the signaling pathways activated at low and high NPY concentrations were similar. The mitogenic effect of the peptide at all doses was completely blocked by inhibitors of calcium/calmodulin-dependent kinase II (CaMKII), protein kinase C (PKC), and mitogen-activated protein kinase kinase, MEK1/2. Thus, in VSMCs, NPY-mediated mitogenesis signals primarily via Y1 receptors activating 2 Ca2+-dependent, growth-promoting pathways -- PKC and CaMKII. At the high-affinity peak, these 2 pathways are amplified by Y5 receptor-mediated, calcium-independent inhibition of the adenylyl cyclase - protein kinase A (PKA) pathway. All 3 mechanisms converge to the extracellular signal-regulated kinases (ERK1/2) signaling cascade and lead to VSMC proliferation.  相似文献   
993.

Background

Various intramammary suspensions containing cloxacillin benzathine are registered for use in cattle as antibiotics for intramammary use at drying off. To ensure antibacterial efficacy, the glandular tissue concentration of an antimicrobial agent must be sufficient. Since the possibilities to measure concentrations in the different areas of the glandular tissue in vivo are very limited, it was the aim of the present study to examine the distribution of cloxacillin in vitro using the isolated perfused bovine udder.

Methods

Mammary glands taken at slaughter from healthy lactating cows were perfused in vitro with warmed and gassed Tyrode solution. 600 mg cloxacillin benzathine were administered as Orbenin Extra Dry Cow by the intramammary route to six front and rear quarters each. Samples of glandular tissue - at different distances from and vertical to the teat right up to the udder base - were gathered from the treated quarters after 6 h. Perfusate was also sampled before and hourly after treatment for 6 h. The cloxacillin content of the tissue samples and perfusate samples was analysed by high performance liquid chromatography.

Results

The concentration of cloxacillin in the glandular tissue of front quarters measured 6 h after administration tended to decrease with increasing vertical distance from the teat. The decrease pattern of the concentration was not quite clear in rear quarters. A considerable variation in the tissue concentrations of cloxacillin was obvious, which reflects in vivo conditions. The concentrations measured in the perfusate samples were below the limit of quantification at all time points, indicating limited absorption of the antibiotic from the glandular tissue.

Conclusion

After intramammary administration of the dry off product containing cloxacillin benzathine concentrations of more than 0.5 μg/g (MIC) were reached in all regions of the front and rear quarters.  相似文献   
994.
Reduced sizes of implantable cardiac pacemakers and clinical advances have led to a higher feasibility of using such devices in younger patients including children. Increased structural demands deriving from reduced device size and more active recipients require detailed knowledge of in vivo mechanical conditions to ensure device reliability. Objective of this study was the proof of feasibility of a system for the measurement of in vivo mechanical loadings on pacemaker implants. The system comprised the following: implantable instrumented pacemaker (IPM) with six force sensors, accelerometer and radio-frequency (RF) transceiver; RF data logging system and video capture system. Three Chacma baboons (20.6±1.15 kg) received one pectoral sub-muscular IPM implant. After wound healing, forces were measured during physical activities. Forces during range of motion of the arm were assessed on the anaesthetized animals prior to device explantation. Mass, volume and dimensions of the excised Pectoralis major muscles were determined after device explantation. Remote IPM activation and data acquisition were reliable in the indoor cage environment with transceiver distances of up to 3 m. Sampling rates of up to 1000 Hz proved sufficient to capture dynamic in vivo loadings. Compressive forces on the IPM in conscious animals reached a maximum of 77.2±54.6 N during physical activity and were 22.2±7.3 N at rest, compared with 34.6±15.7 N maximum during range of motion and 13.4±3.3 N at rest in anaesthetized animals. The study demonstrated the feasibility of the developed system for the assessment of in vivo mechanical loading conditions of implantable pacemakers with potential for use for other implantable therapeutic devices.  相似文献   
995.
The human pathogen Streptococcus pneumoniae is one of the main causative agents of respiratory tract infections. At present, clinical isolates of S. pneumoniae often exhibit decreased susceptibility toward beta-lactams, a phenomenon linked to multiple mutations within the penicillin-binding proteins (PBPs). PBP2x, one of the six PBPs of S. pneumoniae, is the first target to be modified under antibiotic pressure. By comparing 89 S. pneumoniae PBP2x sequences from clinical and public data bases, we have identified one major group of sequences from drug-sensitive strains as well as two distinct groups from drug-resistant strains. The first group includes proteins that display high similarity to PBP2x from the well characterized resistant strain Sp328. The second group includes sequences in which a signature mutation, Q552E, is found adjacent to the third catalytic motif. In this work, a PBP2x from a representative strain from the latter group (S. pneumoniae 5259) was biochemically and structurally characterized. Phenotypical analyses of transformed pneumococci show that the Q552E substitution is responsible for most of the reduction of strain susceptibility toward beta-lactams. The crystal structure of 5259-PBP2x reveals a change in polarity and charge distribution around the active site cavity, as well as rearrangement of strand beta3, emulating structural changes observed for other PBPs that confer drug resistance to Gram-positive pathogens. Interestingly, the active site of 5259-PBP2x is in closed conformation, whereas that of Sp328-PBP2x is open. Consequently, S. pneumoniae has evolved to employ the same protein in two distinct mechanisms of antibiotic resistance.  相似文献   
996.
Summary Plant cells are sensitive to the antibiotic bleomycin, a DNA damaging glycopeptide. A bleomycin resistance determinant, located on transposon Tn5 and functional in bacteria, has been cloned in a plant expression vector and introduced into Nicotiana plumbaginifolia using Agrobacterium tumefaciens. The expression of this determinant in plant cells confers resistance to bleomycin and allows selection of transformed plant cells.  相似文献   
997.
Genetic diversity of crop species in sub-Sahelian Africa is still poorly documented. Among such crops, pearl millet is one of the most important staple species. In Niger, pearl millet covers more than 65% of the total cultivated area. Analyzing pearl millet genetic diversity, its origin and its dynamics is important for in situ and ex situ germplasm conservation and to increase knowledge useful for breeding programs. We developed new genetic markers and a high-throughput technique for the genetic analysis of pearl millet. Using 25 microsatellite markers, we analyzed genetic diversity in 46 wild and 421 cultivated accessions of pearl millet in Niger. We showed a significantly lower number of alleles and lower gene diversity in cultivated pearl millet accessions than in wild accessions. This result contrasts with a previous study using iso-enzyme markers showing similar genetic diversity between cultivated and wild pearl millet populations. We found a strong differentiation between the cultivated and wild groups in Niger. Analyses of introgressions between cultivated and wild accessions showed modest but statistically supported evidence of introgressions. Wild accessions in the central region of Niger showed introgressions of cultivated alleles. Accessions of cultivated pearl millet showed introgressions of wild alleles in the western, central, and eastern parts of Niger.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Cedric Mariac and Viviane Luong have contributed equally to this work.  相似文献   
998.
To develop conducting organic polymers (COPs) as luminescent sensors for determination of toxic heavy metals, a new benzene sulfonic acid‐doped polypyrrole (PPy‐BSA) thin film was electrochemically prepared by cyclic voltammetry (CV) on flexible indium tin oxide (ITO) electrode in aqueous solution. PPy‐BSA film was characterized by FTIR spectrometry, X‐ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). The optical properties of PPy‐BSA were investigated by ultraviolet (UV)‐visible absorption and fluorescence spectrometry in dimethylsulfoxide (DMSO) diluted solutions. PPy‐BSA fluorescence spectra were strongly quenched upon increasing copper(II) ion (Cu2+) and lead(II) ion (Pb2+) concentrations in aqueous medium, and linear Stern–Volmer relationships were obtained, which indicated the existence of a main dynamic fluorescence quenching mechanism. BSA‐PPy sensor showed a high sensitivity for detection of both metallic ions, Cu2+ and Pb2+, with very low limit of detection values of 3.1 and 18.0 nM, respectively. The proposed quenching‐fluorimetric sensor might be applied to the determination of traces of toxic heavy metallic ions in water samples.  相似文献   
999.
Aim To investigate the effects of human‐induced landscape changes in Mediterranean islands on the ecological and evolutionary responses of bird communities and populations. The combination of mass extinction of large mammals and massive deforestation by humans was hypothesized to produce new selection regimes to which organisms were likely to respond. Habitat selection and niche breadth have been investigated at the scale of species, and phenotypic variation at the scale of local populations. Location The study was carried out along habitat gradients and in habitat mosaics at different spatial scales on the island of Corsica and in areas of similar size and structure in continental France. Methods Two sets of gradients have been used for investigating habitat selection and niche breadth: gradients of altitude, and gradients of vegetation structure. Population studies focused on the blue tit, Cyanistes caeruleus. Large samples of breeding attempts by this species in 10 habitats provided detailed data on phenotypic variation of fitness‐related traits both on Corsica and on the mainland. Results The extent of niche space used by birds differed substantially depending on which habitat gradient was considered. Many species have been found to contract their habitat niche along the elevation gradient on Corsica compared with the mainland, whereas all species in the vegetation gradient broadened their niche on the island. Breeding patterns of the blue tit differed considerably depending on whether they settle in deciduous oaks (Quercus humilis) or in evergreen sclerophyllous oaks (Quercus ilex). Phenotypic variation of breeding traits was much higher on the island, where more populations were correctly timed for the best breeding period than on the mainland, a pattern that is likely to result from lower dispersal of organisms on the island. Main conclusions The differences in observed niche breadth between the two series of habitat gradients is explained both by the species‐specific ecology of the species and the human‐induced environmental history of Corsica. Large‐scale landscape changes provided new opportunities for island colonization by non‐forest species, which are isolated as small, ‘fugitive’ local populations. In both gradients, forest species that are typical components of the Corsican bird fauna definitely expanded their niche and occupied a wider range of habitats on Corsica than on the mainland. At the population scale, landscapes included habitat patches with contrasted selection regimes, which resulted in high phenotypic variation for many fitness‐related traits. Reduced dispersal of birds on the island resulted in a much higher degree of local differentiation on Corsica than on the mainland.  相似文献   
1000.
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