Effects of adenosine 3′ : 5′-monophosphate and guanosine 3′ : 5′-monophosphate on calcium uptake and phosphorylation in membrane fractions of vascular smooth muscle

The effects of adenosine 3′ : 5′-monophosphate (cyclic AMP), guanosine 3′ : 5′-monophosphate (cyclic GMP) and exogenous protein kinase on Ca uptake and membrane phosphorylation were studied in subcellular fractions of vascular smooth muscle from rabbit aorta. Two functionally distinct fractions were separated on a continuous sucrose gradient: a light fraction enriched in endoplasmic reticulum (fraction E) and a heavier fraction containing mainly plasma membranes (fraction P).While cyclic AMP and cyclic GMP had no effect on Ca uptake in the absence of oxalate, both cyclic nucleotides inhibited the rate of oxalate-activated Ca uptake when used at concentrations higher than 10^?5 M. The addition of bovine heart protein kinase to either fraction produced an increase in the rate of oxalate-activated Ca uptake which was further augmented by cyclic AMP. Cyclic GMP caused smaller stimulations of protein kinase-catalyzed Ca uptake than cyclic AMP.Mg-dependent phosphorylation, attributable to endogenous protein kinase(s), was inhibited in fraction E by low concentrations (10^?8 M) of both cyclic AMP and cyclic GMP. In fraction P, an inhibition by cyclic AMP occurred also at a concentration of 10^?8 M, while with cyclic AMP a concentration of 10^?5 M was required for a similar inhibition. Bovine heart protein kinase stimulated the phosphorylation of the membrane fractions much more than Ca uptake. In fraction E, in the presence of bovine protein kinase, both cyclic AMP and cyclic GMP stimulated phosphorylation up to 200%. Under these conditions, no stimulation was observed in fraction P.These results are compatible with the hypothesis that in vascular smooth muscle soluble rather than particulate protein kinases are involved in the regulation of intracellular Ca concentration.     

Improved strategy for biosensor-based monitoring of water bodies with diverse organic carbon levels

To protect water resources and to control the water quality it is necessary to develop fast, sensitive, cost-effective, and easy-to-use analytical systems, which are able to measure a variety of contaminants in water. Monitoring water bodies with various matrices can be very difficult. The diverse organic carbon level in water samples (e.g. river water or seawater) causes problems at common analysis and in particular at immunological methods. Here, we demonstrate a new method to overcome the partly occurring matrix problems at quasi-continuous real-world biosensor monitoring. Therefore, we developed an easy matrix referencing method for our fully automated immunoassays that could be adapted to other applications depending on a similar test-format. The method was developed using a synthetic organic carbon standard, and validated using a diluted turf extract. Results for the ultra-sensitive immunoassay for estrone quantification are shown as example. The developed method was verified using immunoassays for testosterone, progesterone, ethinylestradiol, estradiol, and estriol.     

Automated water analyser computer supported system (AWACSS) Part I: Project objectives, basic technology, immunoassay development, software design and networking

A novel analytical system AWACSS (automated water analyser computer-supported system) based on immunochemical technology has been developed that can measure several organic pollutants at low nanogram per litre level in a single few-minutes analysis without any prior sample pre-concentration nor pre-treatment steps. Having in mind actual needs of water-sector managers related to the implementation of the Drinking Water Directive (DWD) (98/83/EC, 1998) and Water Framework Directive WFD (2000/60/EC, 2000), drinking, ground, surface, and waste waters were major media used for the evaluation of the system performance. The instrument was equipped with remote control and surveillance facilities. The system's software allows for the internet-based networking between the measurement and control stations, global management, trend analysis, and early-warning applications. The experience of water laboratories has been utilised at the design of the instrument's hardware and software in order to make the system rugged and user-friendly. Several market surveys were conducted during the project to assess the applicability of the final system. A web-based AWACSS database was created for automated evaluation and storage of the obtained data in a format compatible with major databases of environmental organic pollutants in Europe. This first part article gives the reader an overview of the aims and scope of the AWACSS project as well as details about basic technology, immunoassays, software, and networking developed and utilised within the research project. The second part article reports on the system performance, first real sample measurements, and an international collaborative trial (inter-laboratory tests) to compare the biosensor with conventional anayltical methods.     

Food habits of the barbastelle bat Barbastella barbastellus

The diet of Barbastella barbastellus was investigated through analysis of droppings collected from three maternity roosts in Germany and Switzerland The results showed a high dominance of moths (Lepidoptera). which accounted for 73-94% of the recovered items by volume Flies (Diptera). ranging in size from blow flies (Calliphoridae) and large crane flies (Tipulidae) to small Nematocera, were the second most important prey items (4-17%) Prey types recovered also included small numbers of Trichoptera, Neuroptera, Homoptera. Hymenoptera. Coleoptera and spiders (Araneae) The diet of the barbastelie differs from that of most other bats in Europe m the predominance of moths and the corresponding virtual absence of dung beetles (Coleoptera, Searabaeidae) and midges (Diptera. Chironomidae) Conservation measures for barbastelles should therefore consider facilitation of the diversity and abundance of moths     

Solution structure of a synthetic peptide corresponding to a receptor binding region of FSH (hFSH-β 33–53)

The receptor binding surface of human follicle-stimulating hormone (hFSH) is mimicked by synthetic peptides corresponding to the hFSH-β chain amino acid sequences 33–53 [Santa-Coloma, T. A., Dattatreyamurty, D., and Reichert, L. E., Jr. (1990),Biochemistry 29, 1194–1200], 81–95 [Santa-Coloma, T. A., and Reichert, L. E., Jr. (1990),J. Biol. Chem. 265, 5037–5042], and the combined sequence (33–53)–(81–95) [Santa-Coloma, T. A., Crabb, J. W., and Reichert, L. E., Jr. (1991),Mol. Cell. Endocrinol. 78, 197–204]. These peptides have been shown to inhibit binding of hFSH to its receptor. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy were used to determine the structure of the first peptide in this series, the 21 amino acid peptide hFSH-β-(33–53), H₂N-YTRDLVYKDPARPKIQKTCTF-COOH. Analysis of CD data indicated the presence of approximately equal amounts of antiparallel β-pleated sheet, turns including a β-turn, “other” structures, and a small amount ofa-helix. The major characteristics of the structure were found to be relatively stable at acidicpH and the predominant effect of increased solvent polarity was a small increase ina-helical content. One- and two-dimensional NMR techniques were used to obtain full proton and carbon signal assignments in aqueous solution atpH 3.1. Analysis of NMR results confirmed the presence of the structural features revealed by CD analysis and provided a detailed picture of the secondary structural elements and global folding pattern in hFSH-β-(33–53). These features included an antiparallel β-sheet (residues 38–51 and 46–48), turns within residues 41–46, and 50–52 (a β-turn) and a small N-terminal helical region comprised of amino acids 34–36. One of the turns is facilitated by prolines 42 and 45. Proline-45 was constrained to thetrans conformation, whereas proline-42 favored thetrans conformer (～70%) over thecis (～30%). Two resonances were observed for the single alanine residue (A-43) sequentially proximal to P-42, but the rest of the structure was minimally affected by the isomerization at proline-42. The major population of molecules, containingtrans-42 andtrans-45 prolines, presented 120 NOEs. Distance geometry calculations with 140 distance constraints and energy minimization refinements were used to derive a moderately well-defined model of the peptide's structure. The hFSH-β-(33–53) structure has a highly polar surface composed of six cationic amino acid (arginie-35, lysine-40, arginine-44, lysine-46, glutamine-48, and lysine-49) and two anionic residues (aspartate-36 and aspartic acid-41). A hydrophobic region in the structure is composed of residues in the antiparallel β-sheet and β-turn which fold to produce a distorted “hairpin.” The structure of this domain, together with the protruding and positively charged region in the vicinity of residues 42–45, may mimic the surface of hFSH that binds to the receptor.     

Combined Analysis of Variation in Core,Accessory and Regulatory Genome Regions Provides a Super-Resolution View into the Evolution of Bacterial Populations

The use of whole-genome phylogenetic analysis has revolutionized our understanding of the evolution and spread of many important bacterial pathogens due to the high resolution view it provides. However, the majority of such analyses do not consider the potential role of accessory genes when inferring evolutionary trajectories. Moreover, the recently discovered importance of the switching of gene regulatory elements suggests that an exhaustive analysis, combining information from core and accessory genes with regulatory elements could provide unparalleled detail of the evolution of a bacterial population. Here we demonstrate this principle by applying it to a worldwide multi-host sample of the important pathogenic E. coli lineage ST131. Our approach reveals the existence of multiple circulating subtypes of the major drug–resistant clade of ST131 and provides the first ever population level evidence of core genome substitutions in gene regulatory regions associated with the acquisition and maintenance of different accessory genome elements.     

Comparable High Rates of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli in Birds of Prey from Germany and Mongolia

Frequent contact with human waste and liquid manure from intensive livestock breeding, and the increased loads of antibiotic-resistant bacteria that result, are believed to be responsible for the high carriage rates of ESBL-producing E. coli found in birds of prey (raptors) in Central Europe. To test this hypothesis against the influence of avian migration, we initiated a comparative analysis of faecal samples from wild birds found in Saxony-Anhalt in Germany and the Gobi-Desert in Mongolia, regions of dissimilar human and livestock population characteristics and agricultural practices. We sampled a total of 281 wild birds, mostly raptors with primarily north-to-south migration routes. We determined antimicrobial resistance, focusing on ESBL production, and unravelled the phylogenetic and clonal relatedness of identified ESBL-producing E. coli isolates using multi-locus sequence typing (MLST) and macrorestriction analyses. Surprisingly, the overall carriage rates (approximately 5%) and the proportion of ESBL-producers among E. coli (Germany: 13.8%, Mongolia: 10.8%) were similar in both regions. Whereas bla _CTX-M-1 predominated among German isolates (100%), bla _CTX-M-9 was the most prevalent in Mongolian isolates (75%). We identified sequence types (STs) that are well known in human and veterinary clinical ESBL-producing E. coli (ST12, ST117, ST167, ST648) and observed clonal relatedness between a Mongolian avian ESBL-E. coli (ST167) and a clinical isolate of the same ST that originated in a hospitalised patient in Europe. Our data suggest the influence of avian migratory species in the transmission of ESBL-producing E. coli and challenge the prevailing assumption that reducing human influence alone invariably leads to lower rates of antimicrobial resistance.