Apomorphine-susceptible and apomorphine-unsusceptible Wistar rats differ in their recovery from stress-induced ulcers

The aim of the present study was to investigate the effects of restraint-in-water-stress on gastric ulcerations in two fundamentally different types of animals: the apomorphine-susceptible (APO-SUS) and apomorphine-unsusceptible (APO-UNSUS) rats. APO-SUS and APO-UNSUS do not only differ in their susceptibility to the dopamine agonist apomorphine, but also in stress-induced release of mesolimbic dopamine and corticosterone. All three factors are known to either predict or be involved in gastric ulceration. The results showed that immediately after the stressor the ulcerations in APO-SUS and APO-UNSUS rats were not line-specific. On the contrary, the recovery from gastric ulceration varied between both types of rat: APO-SUS rats did not show any sign of recovery after 6 hours whereas APO-UNSUS rats significantly recovered during the period of 0-6 hr after the stressor. It is hypothesised that this difference is due to the fact that APO-UNSUS rats are characterised by a less and shorter-lasting stress-induced increase of corticosterone. This study provides evidence that the pathological effects of exposure to stressors significantly differ between APO-SUS and APO-UNSUS rats and that genetic factors may direct the process of recovering from ulcers.     

Limited pollen dispersal and biparental inbreeding in Symphonia globulifera in French Guiana

In this paper, we report a study of the mating system and gene flow of Symphonia globulifera, a hermaphroditic, mainly bird-pollinated tree species with a large geographic distribution in the tropical Americas and Africa. Using three microsatellites, we analysed 534 seeds of 28 open pollinated families and 164 adults at the experimental site 'Paracou' in French Guiana. We observed, compared to other tropical tree species, relatively high values for the effective number of alleles. Significant spatial genetic structure was detected, with trees at distances up to 150 m more genetically similar than expected at random. We estimated parameters of the mating system and gene flow by using the mixed mating model and the TwoGener approach. The estimated multilocus outcrossing rate, tm, was 0.920. A significant level of biparental inbreeding and a high proportion of full-sibs were estimated for the 28 seed arrays. We estimated mean pollen dispersal distances between 27 and 53 m according to the dispersal models used. Although the adult population density of S. globulifera in Paracou was relatively high, the joint estimation of pollen dispersal and density of reproductive trees gave effective density estimates of 1.6 and 1.3 trees/ha. The parameters of the mating system and gene flow are discussed in the context of spatial genetic and demographic structures, flowering phenology and pollinator composition and behaviour.     

Inactivation of Ceramide Synthase 6 in Mice Results in an Altered Sphingolipid Metabolism and Behavioral Abnormalities

The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for β-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is ∼35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain.     

Proposed criteria for specific and non-specific chromosomal genotoxicity based on hydrophobic interactions

Tests for chromosomal damage are indispensable in the genotoxicity testing battery. Thus, positive results of clastogenicity or aneugenicity tests are of key relevance in safety assessment and product development. Schultz and Onfelt [N. Schultz, A. Onfelt, Sensitivity of cytokinesis to hydrophobic interactions. Chemical induction of bi- and multi-nucleated cells, Chem. Biol. Interact. 126 (2000) 97-123.] have studied the chemical induction of bi- and multi-nucleated cells in Chinese hamster V79 cells and compared non-specific agents with inducers acting through a known specific mechanism. They separated compounds with a specific action from those with a non-specific action based on lipophilicity, following a theory of hydrophobic interactions with processes of cytokinesis. It appeared possible to broaden the original database of this concept to include aneugenic as well as clastogenic compounds studied in the micronucleus (MN) test. The datasets used for this purpose were (A) the original dataset of Schultz and Onfelt [N. Schultz, A. Onfelt, Sensitivity of cytokinesis to hydrophobic interactions. Chemical induction of bi- and multi-nucleated cells, Chem. Biol. Interact. 126 (2000) 97-123.], and two sets (B, C) of our own data from studies in V79 cells in vitro. As the particular endpoints used were different (A: counts of bi- and multi-nucleated cells, B/C: micronucleus counts) the coherence of the experimental data sets was validated by including compounds belonging to both collections. Data set B included compounds with a specific effect on the mitotic spindle (nitrobenzene and benzonitrile) and data set C included the phytoestrogens genistein and daidzein, as well as a number of hormonal steroids with unknown mode of action. Taking all three data sets (A, B, C) together, the 33 compounds investigated covered a total lipophilicity range of logP between -0.51 (diamide) and 5.65 (17alpha-propylmesterolone). In order to separate statistical outliers (with a specific mode of action to be likely) from the large cluster of compounds with non-specific genotoxicity related to hydrophobic interactions, the method of robust regression was applied. It appeared that all compounds with a specific mode of action were in fact outliers of the lipophilicity rule. Genistein, a weak clastogen causing chromosomal aberrations and being discussed to induce topoisomerase-2 mediated DNA breaks, came close to the statistical borderline between compounds with specific and non-specific chromosomal genotoxicity. A general procedure is proposed, applicable in chemical product development, to screen specific and non-specific modes of action.     

Biomonitoring of ochratoxin A in grain workers

Handling agricultural commodities such as grain can result in an inhalation of mycotoxin-containing dusts. Ochratoxin A (OTA) is particularly well suited for biomonitoring studies due to its long half-life in blood, and served as a marker toxin to investigate whether or not exposure to dusts in occupational contexts may result in elevated OTA blood serum levels. OTA analysis was performed for blood samples (n=61) obtained from a cohort of male workers employed at granaries of several grain handling companies in Germany. OTA was analyzed in plasma extracts by HPLC with fluorimetric detection; calibration curves were run for each batch of samples collected between July 2005 and March 2006, and the level of detection was 0.05 ng/ml plasma. The OTA plasma levels of the 61 grain workers ranged between 0.07 ng/ml and 0.75 ng/ml. The mean (0.28±0.13 ng/ml) and median (0.26 ng/ml) OTA value for this cohort was similar to average values previously reported for the German population. Our results gave no indication that OTA in excess of those originating from typical dietary sources was ingested by these workers. Although measurable OTA concentrations have been found in dust samples collected at the corresponding workplaces (Mayeret al, this issue), the biomonitoring data do not provide evidence for a significant inhalatory burden of OTA in grain workers. Since deoxynivalenol and zearalenone were also detected in the dust samples in concentrations much higher than that of OTA, additional research should try to assess the potential relevance of an inhalation exposure to these mycotoxins. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Loss of FADS2 Function Severely Impairs the Use of HeLa Cells as an In Vitro Model for Host Response Studies Involving Fatty Acid Effects

Scope

Established epithelial cell lines equipped with pattern recognition receptors such as the Toll-like receptor (TLR)-2 are common tools for immune response studies on invading pathogens, e.g. the obligate intracellular species of Chlamydia. Moreover, such models are widely used to elucidate fatty acid-mediated immune effects. In several transformed cell lines, however, unusual loss of metabolic functions was described. The cell lines A549 and HeLa are poorly characterized in this respect. Therefore, we comparatively assessed the metabolic capacity of A549 and HeLa prior to proposed application as in vitro model for fatty acid effects on chlamydial infection.

Methodology/Principal Findings

We incubated both cell lines either with substrates (C18∶2n−6 or C18∶3n−3) or products (C18∶3n−6, C18∶4n−3) of fatty acid desaturase-2 (FADS2), and analysed the fatty acid profiles after 24 h and 72 h by gas chromatography. Based on these data, we suspected that the complete discontinuation of normal biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) in HeLa was due to loss of FADS2 function. Consequently, prostaglandin E2 (PGE2) formation was less inducible by TLR2 stimulation in HeLa, likely as a result of not only insufficient supply of precursors but also weak cyclooxygenase-2 (COX-2) response. In accordance, Chlamydia infection rates were consistently lower in HeLa than in A549. Sequence analysis revealed no alteration within the FADS2 gene in HeLa. The FADS2 expression level, however, was significantly lower and, in contrast to A549, not regulated by C18∶2n−6. A549 exhibited regular fatty acid metabolism and enzyme functionality.

Conclusions/Significance

Our data show that HeLa cells considerably differ from A549 at several stages of fatty acid metabolism. The poor metabolic potential of HeLa, mainly concerning FADS2 upstream of COX-2 function, calls into question whether these cells represent a good model to unveil fatty acid or downstream eicosanoid effects in the course of intracellular bacterial infection.     

Identification of proteins related to the stress response in <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> V583 caused by bovine bile

Background  

Enterococcus faecalis is an opportunistic pathogen and one of the most important causes of hospital infections. Bile acids are a major stress factor bacteria have to cope with in order to colonize and survive in the gastro-intestinal tract. The aim of this study was to investigate the effects of bile acids on the intracellular proteome of E. faecalis V583.     

Genetic variation for growth,morphological, and physiological traits in a wild population of the Neotropical shade­tolerant rainforest tree <Emphasis Type="BoldItalic">Sextonia rubra</Emphasis> (Mez) van der Werff (Lauraceae)

Quantitative genetic diversity is a fundamental component of the interaction between natural populations and their environment. In breeding programmes, quantitative genetic studies on tropical trees have so far focused on fast-growing, light-demanding species, but no information exists on shade-tolerant, slow-growing species. For this study, 27 3-year-old open-pollinated families of the Neotropical shade-tolerant rainforest tree Sextonia rubra were measured in semicontrolled conditions for 20 morphological, growth, and photosynthesis traits; the effect of genetic relatedness, habitat of provenance, and mother tree status on seedling traits was analysed. Nine traits displayed significant genetic effects, while mother tree status and habitat effects were not significant (P > 0.05) for an y trait. Estimated heritability varied between 0.14 and 0.28, with growth-related traits having the highest values. Additive genetic variation correlated positively with nonheritable variation, suggesting that ecological–evolutionary factors increasing or decreasing additive genetic variance may also affect nonheritable variation in the same direction. Our results suggest that quantitative genetic variability should be taken into account in ecological studies on, and in the management of, natural tropical rainforests; further research is needed to investigate genetic × environment interactions, in particular from the point of view of the genetic response of shade-tolerant plant species to variations in light availability.     

Determination of a new antimalarial drug, benflumetol, in blood plasma by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic assay for determination of a new antimalarial drug (benflumetol, BFL) is described. After extraction with hexane-diethyl ether (70:30, v/v) from plasma, BFL was analysed using a C₁₈ Partisil 10 ODS-3 reversed-phase stainless steel column and a mobile phase of acetonitrile-0.1 M ammonium acetate (90:10, v/v) adjusted to pH 4.9 with ultraviolet detection at 335 nm. The mean recovery of BFL over a concentration range of 50–400 ng/ml was 96.8±5.2%. The within-day and day-to-day coefficients of variation were 1.8–4.0 and 1.8–4.2%, respectively. The minimum detectable concentration in plasma for BFL was 5 ng/ml with a C.V. of less than 10%. This method was found to be suitable for clinical pharmacokinetic studies.