Cholinesterases preceding major tracts in vertebrate neurogenesis

The role of acetylcholinesterase (AChE) in neurotransmission is well known. But long before synapses are formed in vertebrates, AChE is expressed in young postmitotic neuroblasts that are about to extend the first long tracts. AChE histochemistry can thus be used to map primary steps of brain differentiation. Preceding and possibly inducing AChE in avian brains, the closely related butyrylcholinesterase (BChE) spatially foreshadows AChE-positive cell areas and the course of their axons. In particular, before spinal motor axons grow, their corresponding rostral sclerotomes and myotomes express BChE, and both their neuronal source and myotomal target cells express AChE. Since axon growth has been found inhibited by acetylcholine, it is postulated that both cholinesterases can attract neurite growth cones by neutralizing the inhibitor. Thus, the early expression of both cholinesterases that is at least partially independent from classical cholinergic synaptogenesis, sheds new light on the developmental and medical significance of these enzymes.     

Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus Nasal Carriage in a Random Sample of Non-Hospitalized Adult Population in Northern Germany

Objective

The findings from truly randomized community-based studies on Staphylococcus aureus nasal colonization are scarce. Therefore we have examined point prevalence and risk factors of S. aureus nasal carriage in a non-hospitalized population of Braunschweig, northern Germany.

Methods

A total of 2026 potential participants were randomly selected through the resident''s registration office and invited by mail. They were requested to collect a nasal swab at home and return it by mail. S. aureus was identified by culture and PCR. Logistic regression was used to determine risk factors of S. aureus carriage.

Results

Among the invitees, 405 individuals agreed to participate and 389 provided complete data which was included in the analysis. The median age of the participants was 49 years (IQR: 39–61) and 61% were females. S. aureus was isolated in 85 (21.9%; 95% CI: 18.0–26.2%) of the samples, five of which were MRSA (1.29%; 95% CI: 0.55–2.98%). In multiple logistic regression, male sex (OR = 3.50; 95% CI: 2.01–6.11) and presence of allergies (OR = 2.43; 95% CI: 1.39–4.24) were found to be associated with S. aureus nasal carriage. Fifty five different spa types were found, that clustered into nine distinct groups. MRSA belonged to the hospital-associated spa types t032 and t025 (corresponds to MLST CC 22), whereas MSSA spa types varied and mostly belonged to spa-CC 012 (corresponds to MLST CC 30), and spa-CC 084 (corresponds to MLST CC 15).

Conclusion

This first point prevalence study of S. aureus in a non-hospitalized population of Germany revealed prevalence, consistent with other European countries and supports previous findings on male sex and allergies as risk factors of S. aureus carriage. The detection of hospital-associated MRSA spa types in the community indicates possible spread of these strains from hospitals into the community.     

Characterization of a mutation that results in independence of oxidosqualene cyclase (Erg7) activity from the downstream 3-ketoreductase (Erg27) in the yeast ergosterol biosynthetic pathway

In yeast, deletion of ERG27, which encodes the sterol biosynthetic enzyme, 3-keto-reductase, results in a concomitant loss of the upstream enzyme, Erg7p, an oxidosqualene cyclase (OSC). However, this phenomenon occurs only in fungi, as mammalian Erg27p orthologues are unable to rescue yeast Erg7p activity. In this study, an erg27 mutant containing the mouse ERG27 orthologue was isolated that was capable of growing without sterol supplementation (FGerg27). GC/MS analysis of this strain showed an accumulation of squalene epoxides, 3-ketosterones, and ergosterol. This strain which was crossed to a wildtype and daughter segregants showed an accumulation of squalene epoxides as well as ergosterol indicating that the mutation entailed a leaky block at ERG7. Upon sequencing the yeast ERG7 gene an A598S alteration was found in a conserved alpha helical region. We theorize that this mutation stabilizes Erg7p in a conformation that mimics Erg27p binding. This mutation, while decreasing OSC activity still retains sufficient residual OSC activity such that the strain in the presence of the mammalian 3-keto reductase enzyme functions and no longer requires the yeast Erg27p. Because sterol biosynthesis occurs in the ER, a fusion protein was synthesized combining Erg7p and Erg28p, a resident ER protein and scaffold of the C-4 demethyation complex. Both FGerg27 and erg27 strains containing this fusion plasmid and the mouse ERG27 orthologue showed restoration of ergosterol biosynthesis with minimal accumulation of squalene epoxides. These results indicate retention of Erg7p in the ER increases its activity and suggest a novel method of regulation of ergosterol biosynthesis.     

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.     

Embryonic chicken retinal cells can regenerate all cell layers in vitro,but ciliary pigmented cells induce their correct polarity

Summary The capacities of retinal and pigmented cells to regenerate histotypic in-vitro-retinae (IVR) in rotary culture were investigated by dividing the eye cups of 6-day-old chicken embryos into a central and a peripheral part; they were cut along the ora serrata, and the retinal and the pigmented constituents of both parts were isolated. The 4 dissociated cell populations were cultured separately and in all double combinations. Two different types of IVR's were generated; one developed from central or peripheral retinal cells, the other required the addition of pigmented cells from the ciliary margin of the eye. The shape of these IVR's was examined using scanning electron microscopy, and they were also characterized histologically. The acetylcholinesterase pattern marked the inner half of the retina; F11-antibody and a peanut agglutinin marker revealed both plexiform layers and a radial fiber system. In both types, organized histotypical areas consisted of complete sets of retinal layers. In the type containing pigmented cells from the eye periphery, the sequence of layers was identical with that of an in-situ-retina (laminar IVR). In IVR's derived from retinal cells only, the sequence of layers was reversed (rosetted IVR).     

Inhibition of cell proliferation by cytosin-arabinoside and its interference with spatial and temporal differentiation patterns in the chick retina

Summary Incubation of chick embryos with 200 nmoles/ egg of cytosine arabinoside (AraC) completely inhibits cell proliferation in the embryo. At an age older than embryonic day 4 (E4) more than 90% of the embryos survive this treatment. The drug induces various malformations; in particular the retina is heavily affected. This simple method offers the possibility to study the effect of a more or less decreased cell production on processes of further differentiation and histogenesis of retinal tissue.The following results are obtained: (1) In spite of the inhibition of cell proliferation in the retina by AraC an abnormally thick basal lamina is found and the expansion of the eye still proceeds, indicating that eye growth is not only dependent on retinal cell numbers. (2) Stereotyped malformations of retinal histogenesis are induced and categorized into three groups: in addition to areas of normal structure cells are found arranged in rosettes and in half-rosettes sometimes linked by areas of undefined transient cell arrangements. The results point to a strong tendency of a severely diminished cell population to form regularly laminated retinal-like structures as long as a minimal ratio of cell types is given. (3) The spatio-temporal appearance of the type of retinal malformation in a given retinal area is dependent on the time of AraC exposure and thus on the degree of differentiation reached at a spatio-temporal spot: Full rosettes develop at earlier, and half-rosettes at later stages of AraC interference. Furthermore, deformities first appear on temporal and ventral sides. Thus, the establishment of these malformations follows and reflects the normal sequence of differentiation within the retina. (4) Cells within rosettes organize in specific layers and start to differentiate normally. This shows that earlier formed cells are not dependent on the influence of factors derived from cells that are formed later for their proper differentiation.Abbreviations AChE acetylcholinesterase - AraC cytosin arabinoside - d'cyt 2 desoxycytidine - FITC-Con A fluorescent concanavalin A - FITC-PNA fluorescent peanut agglutinin - GCL ganglion cell layer - INL inner nuclear layer - IPL inner plexiform layer - LY Lucifer Yellow Recipient of a grant from the Alexander von Humboldt-Stiftung