NADPH-diaphorase-positive nerve fibers associated with motor endplates in the rat esophagus: new evidence for co-innervation of striated muscle by enteric neurons

NADPH-diaphorase histochemistry was combined with demonstration of acetylcholinesterase and immunocytochemistry for calcitonin gene-related peptide to study esophageal innervation in the rat. Most of the myenteric neurons stained positively for NADPH-diaphorase, as did numerous varicose nerve fibers in the myenteric plexus, among striated muscle fibers, around arterial blood vessels, and in the muscularis mucosae. A majority of motor endplates (as demonstrated by acetylcholinesterase histochemistry or calcitonin gene-related peptide immunocytochemistry) were associated with fine varicose NADPH-diaphorase-positive nerve fibers. Analysis of brainstem nuclei, sensory vagal, spinal, and sympathetic ganglia in normal and neonatally capsaicin-treated rats, and comparison with anterogradely labeled vagal branchiomotor, preganglionic and sensory fibers led to the conclusion that NADPH-diaphorase-positive fibers on motor endplates originate in esophageal myenteric neurons. No association of NADPH-diaphorasepositive nerve fibers with motor endplates was found in other organs containing striated muscle. These results suggest extensive, presumably nitrergic, co-innervation of esophageal striated muscle fibers by enteric neurons. Thus, control of peristalsis in the esophagus of the rat may be more complex than hitherto assumed.     

Comparison of microsatellite and single nucleotide polymorphism markers for the genetic analysis of a Galloway cattle population

Highly informative genetic markers are essential for efficient management of cattle populations, as well as for food safety. After a decade of domination by microsatellite markers, a new type of genetic marker, single nucleotide polymorphism (SNP), has recently appeared on the scene. In the present study, the exclusion power of both kinds of markers with regards to individual identification and parental analysis was directly compared in a Galloway cattle population. Seventeen bovine microsatellites were distributed in three incremental marker sets (10, 14 and 17 microsatellite markers) and used for cattle genotyping. A set of 43 bovine SNP was used for genotyping the same cattle population. The accuracy of both kinds of markers in individual identification was evaluated using probability of identity estimations. These were 2.4 x 10(-8) for the 10 microsatellite set, 2.3 x 10(-11) for the 14 microsatellite set, and 1.4 x 10(-13) for the 17 microsatellite marker set. For the 43 SNP markers, the estimated probability of identity was 5.3 x 10(-11). The exclusion power of both kinds of markers in parental analysis was evaluated using paternity exclusion estimations, and, in addition to this, by estimation of the parental exclusion probability in 18 Galloway family trios. Paternity exclusion was estimated to be over 99% for microsatellites, and approx. 98% for SNP. Both, microsatellite and SNP sets of markers showed similar parental exclusion probabilities.     

Cancer of the endometrium: current aspects of diagnostics and treatment

Background  

Endometrial cancer represents a tumor entity with a great variation in its incidence throughout the world (range 1 to 25). This suggests enormous possibilities of cancer prevention due to the fact that the incidence is very much endocrine-related, chiefly with obesity, and thus most frequent in the developed world. As far as treatment is concerned, it is generally accepted that surgery represents the first choice of treatment. However, several recommendations seem reasonable especially with lymphadenectomy, even though they are not based on evidence. All high-risk cases are generally recommended for radiotherapy.     

Acetylcholine Receptor (AChR) Clustering Is Regulated Both by Glycogen Synthase Kinase 3β (GSK3β)-dependent Phosphorylation and the Level of CLIP-associated Protein 2 (CLASP2) Mediating the Capture of Microtubule Plus-ends

The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2–9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2–8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends.     

On-farm experiments over 5 years in a grain maize/winter wheat rotation: effect of maize residue treatments on <Emphasis Type="Italic">Fusarium graminearum</Emphasis> infection and deoxynivalenol contamination in wheat

Over the course of 5 years, different maize residue treatments were conducted on 14 zero tillage on-farm sites in Switzerland to evaluate their effect on the development of Fusarium head blight (FHB) and the contamination with the mycotoxin deoxynivalenol (DON) in winter wheat grains and wheat straw following grain maize. Two experimental series with three and five different treatments were carried out, respectively. Fusarium graminearum (Schwabe) was the predominant FHB-causing species with an overall incidence of 15% infected wheat grains. A significant correlation between symptoms in the field, F. graminearum incidence and DON content in wheat grains and wheat straw was observed. The average DON content in both wheat grains and wheat straw was approximately 5,000 μg/kg and thus several times higher than the European maximum limit of 1,250 μg/kg for unprocessed small-grain cereals for human consumption. Of all grain samples, 74% were above the maximum limit. Pooled over both experimental series, the average reduction of DON in grains through treatments of the maize residue compared with a control treatment ranged between 21 and 38%. The effect of various other factors, including the year, the wheat variety, the site, the maize hybrid and the production system was evaluated as well. The year and the wheat variety were the most important FHB influencing factors. Over all treatments, the variety Levis showed a fivefold higher average DON content compared with the variety Titlis. From different categories of maize residue particles, intact pieces of 5–15 cm length were strongly correlated with F. graminearum incidence and DON content in grains. During the time course of this study, the recommendation from a preliminary version of the internet-based DON forecasting system FusaProg to apply or to omit a fungicide treatment was correct in 32 out of 42 cases. The results are currently being used to optimise the FusaProg models. This study has shown that in a grain maize/winter wheat rotation, the DON content in wheat grains frequently exceeded the European maximum limit, even with a thorough treatment of maize residues and less susceptible wheat varieties. Hence, in order to reduce the contamination risk in a zero tillage system, the crop rotation needs to be modified.     

A benzo[a]pyrene metabolite-DNA adduct occurring in the activating cell but not in exogenous DNA

[3H]Benzo[a]pyrene (BP) and salmon sperm DNA were incubated with hepatocytes from 5,6-benzo-flavone-treated rats. The cellular DNA and the exogenously added DNA were separately isolated, hydrolyzed and chromatographed on a Sephadex LH-20 column. The extracellular DNA yielded 3 peaks of radioactivity in the chromatographic eluate. The cellular DNA contained an additional peak suggesting the formation of a DNA adduct from a metabolite that does not leave the cell.     

Strategy for genotoxicity testing--metabolic considerations

The report from the 2002 International Workshop on Genotoxicity Tests (IWGT) Strategy Expert Group emphasized metabolic considerations as an important area to address in developing a common strategy for genotoxicity testing. A working group convened at the 2005 4th IWGT to discuss this area further and propose practical strategy recommendations. To propose a strategy, the working group reviewed: (1) the current status and deficiencies, including examples of carcinogens "missed" in genotoxicity testing, established shortcomings of the standard in vitro induced S9 activation system and drug metabolite case examples; (2) the current status of possible remedies, including alternative S9 sources, other external metabolism systems or genetically engineered test systems; (3) any existing positions or guidance. The working group established consensus principles to guide strategy development. Thus, a human metabolite of interest should be represented in genotoxicity and carcinogenicity testing, including evaluation of alternative genotoxicity in vitro metabolic activation or test systems, and the selection of a carcinogenicity test species showing appropriate biotransformation. Appropriate action triggers need to be defined based on the extent of human exposure, considering any structural knowledge of the metabolite, and when genotoxicity is observed upon in vitro testing in the presence of metabolic activation. These triggers also need to be considered in defining the timing of human pharmaceutical ADME assessments. The working group proposed two strategies to consider; a more proactive approach, which emphasizes early metabolism predictions to drive appropriate hazard assessment; and a retroactive approach to manage safety risks of a unique or "major" metabolite once identified and quantitated from human clinical ADME studies. In both strategies, the assessment of the genotoxic potential of a metabolite could include the use of an alternative or optimized in vitro metabolic activation system, or direct testing of an isolated or synthesized metabolite. The working group also identified specific areas where more data or experiences need to be gained to reach consensus. These included defining a discrete exposure action trigger for safety assessment and when direct testing of a metabolite of interest is warranted versus the use of an alternative in vitro activation system, a universal recommendation for the timing of human ADME studies for drug candidates and the positioning of metabolite structural knowledge (through in silico systems, literature, expert analysis) in supporting metabolite safety qualification. Lastly, the working group outlined future considerations for refining the initially proposed strategies. These included the need for further evaluation of the current in vitro genotoxicity testing protocols that can potentially perturb or reduce the level of metabolic activity (potential alterations in metabolism associated with both the use of some solvents to solubilize test chemicals and testing to the guidance limit dose), and proposing broader evaluations of alternative metabolic activation sources or engineered test systems to further challenge the suitability of (or replace) the current induced liver S9 activation source.