Modulation of electrically evoked serotonin release in cultured rat raphe neurons

Electrically evoked release of serotonin (5-HT) and its modulation via 5-HT autoreceptors and alpha(2)-heteroreceptors was studied in primary cell cultures prepared from the embryonic (ED 15) rat mesencephalic brain region comprising the raphe nuclei. Cultures were grown for up to 3 weeks on circular glass coverslips. They developed a dense network of non-neuronal and neuronal cells, some of which were positive for tryptophan hydroxylase. To measure 5-HT release, the cultures were pre-incubated with [(3)H]5-HT (in the presence of the selective noradrenaline reuptake inhibitor oxaprotiline [1 micromol/L]), superfused with modified Krebs-Henseleit medium containing 6-nitroqipazine [1 micromol/L] and electrically stimulated using two conditions. Condition A: 360 pulses, 3 Hz, 0.5 ms, 90 mA, or condition B: 4 pulses 100 Hz, 0.5 ms, 90 mA (a condition which diminishes interactions with endogenously released transmitters during ongoing stimulation). After only 1 week in culture, the electrically evoked overflow of [(3)H] was Ca(2+) dependent and tetrodotoxin sensitive, suggesting an action-potential-induced exocytotic release of 5-HT. Using stimulation condition A in cultures grown for 2 weeks, both basal and evoked 5-HT release were strongly enhanced by methiotepine (1 micromol/L) but unaffected by the 5-HT(1B) autoreceptor agonist CP-93, 129 (1 micromol/L) and the alpha(2)-adrenoceptor agonist UK-14, 304 (1 micromol/L). Conversely, using stimulation condition B, not only CP-93, 129 (IC(50) 8.1 +/- 1.4 nmol/L) and UK-14, 304 (IC(50) 14.9 +/- 1.6 nmol/L) had inhibitory effects on cells grown for 2 weeks, but also the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin. In conclusion, we describe for the first time electrically evoked release of 5-HT from primary cultures of fetal raphe cells and its modulation via 5-HT(1B) and 5-HT(1A) auto- and alpha(2)-heteroreceptors. Such cultured raphe cells may represent a suitable model to study expression and development of presynaptic receptors on serotonergic neurons in-vitro.     

Knockdown and mortality comparisons among spinosad-, imidacloprid-, and methomyl-containing baits against susceptible Musca domestica (Diptera: Muscidae) under laboratory conditions

The activity of spinosad, imidacloprid, and methomyl baits and technical actives were assessed against susceptible house flies, Musca domestica L. (Diptera: Muscidae). In a feeding assay, imidacloprid affected flies more rapidly than methomyl or spinosad, but spinosad was 2.7 times more potent than methomyl and 8 times more potent than imidacloprid. The profile of technical actives correlated with their respective fly bait formulations in laboratory assays. Although having the most rapid onset of activity in laboratory tests, up to 50% of flies remained alive after exposure to imidacloprid bait. In contrast, <5% of flies survived 24-h exposure to spinosad or methomyl baits. High temperature reduced the knockdown activity of imidacloprid bait and slowed the speed of kill for spinosad and methomyl baits over a 24-h exposure period. Spinosad and methomyl baits were also superior to imidacloprid when applied to the floors of environmentally controlled rooms at label recommended rates, providing good fly control for up to 21 d. The fact that a significant percentage of flies exposed to imidacloprid were rapidly knocked down but subsequently remained alive in all of the assays suggested that flies were recovering from initial exposure to this compound. Given its favorable safety profile, a high degree of initial and residual activity comparable with methomyl and lack of cross-resistance to other chemistries, spinosad bait may be a valuable component of house fly control programs to help control or delay the emergence of resistant populations.     

Gene and genon concept: coding versus regulation

We analyse here the definition of the gene in order to distinguish, on the basis of modern insight in molecular biology, what the gene is coding for, namely a specific polypeptide, and how its expression is realized and controlled. Before the coding role of the DNA was discovered, a gene was identified with a specific phenotypic trait, from Mendel through Morgan up to Benzer. Subsequently, however, molecular biologists ventured to define a gene at the level of the DNA sequence in terms of coding. As is becoming ever more evident, the relations between information stored at DNA level and functional products are very intricate, and the regulatory aspects are as important and essential as the information coding for products. This approach led, thus, to a conceptual hybrid that confused coding, regulation and functional aspects. In this essay, we develop a definition of the gene that once again starts from the functional aspect. A cellular function can be represented by a polypeptide or an RNA. In the case of the polypeptide, its biochemical identity is determined by the mRNA prior to translation, and that is where we locate the gene. The steps from specific, but possibly separated sequence fragments at DNA level to that final mRNA then can be analysed in terms of regulation. For that purpose, we coin the new term “genon”. In that manner, we can clearly separate product and regulative information while keeping the fundamental relation between coding and function without the need to introduce a conceptual hybrid. In mRNA, the program regulating the expression of a gene is superimposed onto and added to the coding sequence in cis - we call it the genon. The complementary external control of a given mRNA by trans-acting factors is incorporated in its transgenon. A consequence of this definition is that, in eukaryotes, the gene is, in most cases, not yet present at DNA level. Rather, it is assembled by RNA processing, including differential splicing, from various pieces, as steered by the genon. It emerges finally as an uninterrupted nucleic acid sequence at mRNA level just prior to translation, in faithful correspondence with the amino acid sequence to be produced as a polypeptide. After translation, the genon has fulfilled its role and expires. The distinction between the protein coding information as materialised in the final polypeptide and the processing information represented by the genon allows us to set up a new information theoretic scheme. The standard sequence information determined by the genetic code expresses the relation between coding sequence and product. Backward analysis asks from which coding region in the DNA a given polypeptide originates. The (more interesting) forward analysis asks in how many polypeptides of how many different types a given DNA segment is expressed. This concerns the control of the expression process for which we have introduced the genon concept. Thus, the information theoretic analysis can capture the complementary aspects of coding and regulation, of gene and genon.     

A Bnr-like formin links actin to the spindle pole body during sporulation in the filamentous fungus Ashbya gossypii

Formin proteins are nucleators of actin filaments and regulators of the microtubule cytoskeleton. As such, they play important roles in the development of yeast and other fungi. We show here that AgBnr2, a homologue of the ScBnr1 formin from the filamentous fungus Ashbya gossypii, localizes to the spindle pole body (SPB), the fungal analogue of the centrosome of metazoans. This protein plays an important role in the development of the typical needle-shaped spores of A. gossypii, as suggested by several findings. First, downregulation of AgBNR2 causes defects in sporangium formation and a decrease in the total spore number. Second, a fusion of AgBNR2 to GFP that is driven by the native AgBNR2 promoter is only visible in sporangia. Third, AgBnr2 interacts with a AgSpo21, a sporulation-specific component of the SPB. Furthermore, we provide evidence that AgBnr2 might nucleate actin cables, which are connected to SPBs during sporulation. Our findings add to our understanding of fungal sporulation, particularly the formation of spores with a complex, elongated morphology, and provide novel insights into formin function.     

Reduction of calcium release site models via moment fitting of phase-type distributions

Models of calcium (Ca(2 +)) release sites derived from continuous-time Markov chain (CTMC) models of intracellular Ca(2 +) channels exhibit collective gating reminiscent of the experimentally observed phenomenon of Ca(2 +) puffs and sparks. In order to overcome the state-space explosion that occurs in compositionally defined Ca(2 +) release site models, we have implemented an automated procedure for model reduction that replaces aggregated states of the full release site model with much simpler CTMCs that have similar within-group phase-type sojourn times and inter-group transitions. Error analysis based on comparison of full and reduced models validates the method when applied to release site models composed of 20 three-state channels that are both activated and inactivated by Ca(2 +). Although inspired by existing techniques for fitting moments of phase-type distributions, the automated reduction method for compositional Ca(2 +) release site models is unique in several respects and novel in this biophysical context.     

Deficits in long-term recognition memory reveal dissociated subtypes in congenital prosopagnosia

The study investigates long-term recognition memory in congenital prosopagnosia (CP), a lifelong impairment in face identification that is present from birth. Previous investigations of processing deficits in CP have mostly relied on short-term recognition tests to estimate the scope and severity of individual deficits. We firstly report on a controlled test of long-term (one year) recognition memory for faces and objects conducted with a large group of participants with CP. Long-term recognition memory is significantly impaired in eight CP participants (CPs). In all but one case, this deficit was selective to faces and didn't extend to intra-class recognition of object stimuli. In a test of famous face recognition, long-term recognition deficits were less pronounced, even after accounting for differences in media consumption between controls and CPs. Secondly, we combined test results on long-term and short-term recognition of faces and objects, and found a large heterogeneity in severity and scope of individual deficits. Analysis of the observed heterogeneity revealed a dissociation of CP into subtypes with a homogeneous phenotypical profile. Thirdly, we found that among CPs self-assessment of real-life difficulties, based on a standardized questionnaire, and experimentally assessed face recognition deficits are strongly correlated. Our results demonstrate that controlled tests of long-term recognition memory are needed to fully assess face recognition deficits in CP. Based on controlled and comprehensive experimental testing, CP can be dissociated into subtypes with a homogeneous phenotypical profile. The CP subtypes identified align with those found in prosopagnosia caused by cortical lesions; they can be interpreted with respect to a hierarchical neural system for face perception.