Mechanism of wine lactone formation: demonstration of stereoselective cyclization and 1,3-hydride shift

The cyclization mechanism of (E)-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid to wine lactone under acidic aqueous conditions was investigated using the two stereoselectively deuterium-labeled precursors (2E,6R,7Z)-[8-2H]-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid and (2E,7E)-(+/-)-[8-2H]-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid. A detailed analysis of the generated wine lactone isomers by enantioselective multidimensional gas chromatography (MDGC)/ion trap tandem mass spectrometry demonstrates that the formation of wine lactone proceeds via a nonenzymatic stereoselective cationic cyclization cascade that includes a 1,3-hydride shift. Usually, such mechanisms are features of cyclization reactions that are catalyzed by terpene cyclases. This nonenzymatic conversion of an acyclic precursor to a bicyclic monoterpene under relevant cationic cyclization conditions has rarely been observed and confirms recent suggestions that the precursor itself can provide the chemical functionality required for specific steps in the cyclization cascade.     

Radial stem growth in response to microclimate and soil moisture in a drought-prone mixed coniferous forest at an inner Alpine site

Dendroclimatological studies in a dry inner Alpine environment (750 m a.s.l.) revealed different growth response of co-occurring coniferous species to climate, which is assumed to be caused by a temporal shift in wood formation among species. The main focus of this study therefore was to monitor intra-annual dynamics of radial increment growth of mature deciduous and evergreen coniferous species (Pinus sylvestris, Larix decidua and Picea abies) during two consecutive years with contrasting climatic conditions. Radial stem growth was continuously followed by band dendrometers and modelled using Gompertz functions to determine time of maximum growth. Histological analyses of tree ring formation allowed determination of temporal dynamics of cambial activity and xylem cell development. Daily fluctuations in stem radius and radial stem increments were extracted from dendrometer traces, and correlations with environmental variables were performed. While a shift in temporal dynamics of radial growth onset and cessation was detected among co-occurring species, intra-annual radial growth peaked synchronously in late May 2011 and early June 2012. Moist atmospheric conditions, i.e. high relative air humidity, low vapour pressure deficit and low air temperature during the main growing period, favoured radial stem increment of all species. Soil water content and soil temperature were not significantly related to radial growth. Although a temporal shift in onset and cessation of wood formation was detected among species, synchronous culmination of radial growth indicates homogenous exogenous and/or endogenous control. The close coupling of radial growth to atmospheric conditions points to the importance of stem water status for intra-annual growth of drought-prone conifers.     

Recent developments in the laboratory diagnosis of chlamydial infections

There are two main approaches to diagnosing infections by Chlamydia and Chlamydophila spp. in mammals and birds. The first involves the direct detection of the agent in tissue or swab samples, while the second involves the serological screening of blood samples for the presence of anti-chlamydial antibodies. Ultimately, the test that is used is dependent on the types of samples that are submitted to the diagnostic laboratory for analysis. The present paper gives an overview on methodologies and technologies used currently in diagnosis of chlamydial infections with emphasis on recently developed tests. The performance characteristics of individual methods, such as the detection of antigen in smears and in pathological samples, the isolation of the pathogen, various antibody detection tests and DNA-based methods utilising conventional and real-time PCR, as well as DNA microarray technology are assessed, and specific advantages and drawbacks are discussed. Further, a combination of a specific real-time PCR assay and a microarray test for chlamydiae is proposed as an alternative reference standard to isolation by cell culture.     

Report on Salmonella isolates submitted to the German National Veterinary Salmonella Reference Laboratory in the year 1999

The National Salmonella Reference Laboratory (NRL-Salm) of the Federal Institute for Health Protection of Consumers and Veterinary Medicine receives putative Salmonella isolates originating from animals, food, feed and the environment for typing. This report summarises the results of the sero- and phagetyping studies. In livestock S. Typhimurium DT 104 with 24% and S. Enteritidis PT 4 with 9% of all isolations predominate like in the human isolates. These sero- and phagetypes are frequently isolated from food as well and consequently can reach the consumer via the food chain.     

Evidence for oxidative stress involved in physiological leaf spot formation in winter and spring barley

ABSTRACT A leaf spot disease with unknown etiology has become more pronounced in spring and winter barley in Germany in recent years. The symptoms are similar to net blotch and Ramularia leaf spots, but the causal agents of these diseases are not identified. The symptom expression varied much on cultivars. Cultivars most affected by the disease of both spring and winter barley showed a significantly higher level of superoxide (O(2) ) production and lipid peroxidation (malondialdehyde), but a lower level of antioxidant potential expressed as superoxide dismutase (SOD) activity, catalase activity, and integral water-soluble antioxidant capacity (ACW) than insensitive cultivars. A high positive correlation between O(2) production and leaf spot development between ear emergence and milk ripeness was established in the most sensitive winter barley cv. Anoa (r(2) = 0.9622) and spring barley cv. Barke (r(2) = 0.9434). Leaf H(2)O(2) levels increased with the severity of leaf spots. The histochemical localization of O(2) and H(2)O(2) in the tissues adjacent to leaf spots indicated that these two active oxygen species (AOS) are involved in the formation of leaf spots. Reduction of symptom severity by applying strobilurin and azole fungicides was always associated with elevated SOD activity and ACW content and suppressed O(2) production. However, peroxidase activities were significantly higher in sensitive cultivars and in more severely affected tissues and decreased by applying fungicides. Thus, it is assumed that a possible genetic mechanism based on the imbalanced AOS metabolism contributes to formation of physiological leaf spots.     

Development of <Emphasis Type="Italic">Peronospora parasitica</Emphasis> epidemics on radish as modelled by the effects of water vapour saturation deficit and temperature

Epidemics of Peronospora parasitica are strongly affected by temperature and air moisture, and the interaction of these factors. Because a significant percentage of radish plants are grown in greenhouses, it may be possible to influence epidemics by altering the greenhouse climate. The objective of this study was to test the hypothesis that epidemics of P. parasitica can be modelled by the effects of air temperature and moisture in the greenhouse. Such a model could then be used to analyse greenhouse climate control strategies with regard to managing downy mildew. Five radish crops were grown under greenhouse conditions with set-points for heating and ventilation intended to obtain favourable conditions for disease development during the first part of the growing cycle. Subsequent to this first phase, unfavourable conditions were set until harvest. Disease incidence was measured once a week until the radishes reached marketable size. In addition, experiments were carried out in growth chambers in which inoculated plants were subjected to air temperatures between 8 and 27°C, and disease incidence and sporulation intensity were measured. Data from these two experiments were then used to estimate model parameters. In this model, the interactions of air temperature (T) and water vapour saturation deficit (SD) were adequately described by a multiplicative relationship. The simulated epidemics by the fitted model were highly correlated with the observed epidemics (r = 0.91, R ² = 0.83, n = 29). Parameter estimates indicated that T of ca. 20°C and SD < 0.03 hPa resulted in the highest rates of disease development and that the rate was zero when SD > 2.0 hPa. Both experimental data and simulations showed that epidemics of P. parasitica can be effectively controlled by managing the greenhouse climate.     

Binding Sites for Ca2+-Channel Effectors and Ryanodine in Periplaneta americana—Possible Targets for New Insecticides

The calcium channel and the ‘calcium release channel’ of muscle membrane of the cockroach Periplaneta americana have been characterized. Biological assays with calcium channel blockers and ryanodine on different insects and acari revealed pronounced insecticidal effects with ryanodine, but not with calcium channel blockers, at concentrations between 0·1 and 300 μg ml⁻¹. Skeletal muscle membranes derived either from the tubular network or from the sarcoplasmatic reticulum of P. americana were characterized with respect to the binding of the dihydropyridine (DHP) [³H]isradipine (PN 200-110), the phenyl-alkylamine [³H]verapamil and the alkaloid [³H]ryanodine. Preliminary binding studies with the benzothiazepine [³H]diltiazem suggest a low-affinity binding site with a IC₅₀ value of 3·3 μM . All binding sites tested were sensitive to treatment with proteinase K. Optimal conditions for binding of the radioligand ryanodine revealed the highest specific binding at pH 8 and at calcium chloride concentrations between 100 and 500 μM . EGTA at 10 μM abolished 95% of the ryanodine binding. Binding studies with calcium channel binding sites revealed a pronounced effect of low Ca²⁺ concentrations on specific isradipine binding, whereas verapamil and diltiazem binding were only reduced by the presence of 200 μM EGTA. With respect to high Ca²⁺ concentrations, specific binding of diltiazem, isradipine and verapamil was reduced by 73, 40 and 20%, respectively, at 5 mM Ca²⁺. Radioligand binding experiments showed high-affinity binding sites for ryanodine and isradipine. K_D values of 0·95 nM (B_max=550 fmol mg⁻¹ protein) and 0·75 nM (B_max=213 fmol mg⁻¹ protein) were determined respectively. A lower-affinity binding site was identified in binding studies with verapamil (K_D=7·4 nM and B_max=27 fmol mg⁻¹ protein). [³H]isradipine displacement studies with several dihydropyridines revealed the following ranking of affinity: nitrendipine>isradipine>Bay K8664≪nicardipine. Displacement of [³H]verapamil binding by effectors of the phenylalkylamine binding site showed that bepridil and S(-)verapamil had the highest affinities of the compounds tested followed by (±)verapamil, nor-methylverapamil and R(+)verapamil.     

Spatial distribution of soil heavy metal concentrations as indicator of pollution sources at Mount Križna (Great Fatra,central Slovakia)

The objective of this study was to test the suitability of a simple approach to identify the direction from where airborne heavy metals reach the study area as indication of their sources. We examined the distribution of heavy metals in soil profiles and along differently exposed transects. Samples were taken from 10 soils derived from the same parent material along N-, S-, and SE-exposed transects at 0—10, 10—20, and 20—40 cm depth and analyzed for total Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn concentrations. The heavy metal concentrations at 0—10 cm were larger than background concentrations in German arable soils except for Cr (Cd: 0.6—1.8 mg kg^—1; Cr: 39—67; Cu: 40—77; Ni: 87—156; Pb: 48—94; Zn: 71—129; Fe: 26—34 g kg^—1; Mn: 1.1—2.4). Decreasing Cd, Cu, Mn, and Pb concentrations with increasing soil depth pointed at atmospheric inputs. Aluminum and Ni concentrations increased with soil depth. Those of Fe, Cr, and Zn did not change with depth indicating that inputs at most equalled leaching losses. The Pb accumulation in the surface layer (i.e. the ratio between the Pb concentrations at 0—10 to those at 20—40 cm depth) was most pronounced at N-exposed sites; Pb obviously reached Mount Križna mainly by long-range transport from N where several industrial agglomerations are located. Substantial Cd, Cu, and Mn accumulations at the S- and SE-exposed sites indicated local sources such as mining near to the study area which probably are also the reason for slight Cr and Zn accumulations in the SE-exposed soils. Based on a principal component analysis of the total concentrations in the topsoils four metal groups may be distinguished: 1. Cr, Ni, Zn; 2. Mn, Cd; 3. Pb (positive loading), Cu (negative loading); 4. Al, Fe, indicating common sources and distribution patterns. The results demonstrate that the spatial distribution of soil heavy metal concentrations can be used as indication of the location of pollution sources.     

In silico prediction of Gallibacterium anatis pan-immunogens

The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in commercial egg-layers, leading to reduced egg production and increased mortality. Unfortunately, widespread multidrug resistance and antigenic diversity makes it difficult to control infections and novel prevention strategies are urgently needed. In this study, a pan-genomic reverse vaccinology (RV) approach was used to identify potential vaccine candidates. Firstly, the genomes of 10 selected Gallibacterium strains were analyzed and proteins selected on the following criteria; predicted surface-exposure or secretion, none or one transmembrane helix (TMH), and presence in six or more of the 10 genomes. In total, 42 proteins were selected. The genes encoding 27 of these proteins were successfully cloned in Escherichia coli and the proteins expressed and purified. To reduce the number of vaccine candidates for in vivo testing, each of the purified recombinant proteins was screened by ELISA for their ability to elicit a significant serological response with serum from chickens that had been infected with G. anatis. Additionally, an in silico prediction of the protective potential was carried out based on a protein property prediction method. Of the 27 proteins, two novel putative immunogens were identified; Gab_1309 and Gab_2312. Moreover, three previously characterized virulence factors; GtxA, FlfA and Gab_2156, were identified. Thus, by combining the pan-genomic RV approach with subsequent in vitro and in silico screening, we have narrowed down the pan-proteome of G. anatis to five vaccine candidates. Importantly, preliminary immunization trials indicated an in vivo protective potential of GtxA-N, FlfA and Gab_1309.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0080-0) contains supplementary material, which is available to authorized users.