Specification of neuronal identities by feedforward combinatorial coding

Neuronal specification is often seen as a multistep process: earlier regulators confer broad neuronal identity and are followed by combinatorial codes specifying neuronal properties unique to specific subtypes. However, it is still unclear whether early regulators are re-deployed in subtype-specific combinatorial codes, and whether early patterning events act to restrict the developmental potential of postmitotic cells. Here, we use the differential peptidergic fate of two lineage-related peptidergic neurons in the Drosophila ventral nerve cord to show how, in a feedforward mechanism, earlier determinants become critical players in later combinatorial codes. Amongst the progeny of neuroblast 5–6 are two peptidergic neurons: one expresses FMRFamide and the other one expresses Nplp1 and the dopamine receptor DopR. We show the HLH gene collier functions at three different levels to progressively restrict neuronal identity in the 5–6 lineage. At the final step, collier is the critical combinatorial factor that differentiates two partially overlapping combinatorial codes that define FMRFamide versus Nplp1/DopR identity. Misexpression experiments reveal that both codes can activate neuropeptide gene expression in vast numbers of neurons. Despite their partially overlapping composition, we find that the codes are remarkably specific, with each code activating only the proper neuropeptide gene. These results indicate that a limited number of regulators may constitute a potent combinatorial code that dictates unique neuronal cell fate, and that such codes show a surprising disregard for many global instructive cues.     

Multi-domain network infrastructure based on P4 programmable devices for Digital Data Marketplaces

Cluster Computing - There are many organizations interested in sharing data with others, and they can do this only if a multi-domain secure platform is available. Such platforms, often referred to...     

Application of a novel analysis to measure the binding of the membrane-translocating peptide penetratin to negatively charged liposomes

The binding of penetratin, a peptide that has been found useful for cellular delivery of large hydrophilic molecules, to negatively charged vesicles was investigated. The surface charge density of the vesicles was varied by mixing zwitterionic dioleoylphosphatidylcholine (DOPC) and negatively charged dioleoylphosphatidylglycerol (DOPG) at various molar ratios. The extent of membrane association was quantified from tryptophan emission spectra recorded during titration of peptide solution with liposomes. A singular value decomposition of the spectral data demonstrated unambiguously that two species, assigned as peptide free in solution and membrane-bound peptide, respectively, account for the spectral data of the titration series. Binding isotherms were then constructed by least-squares projection of the titration spectra on reference spectra of free and membrane-bound peptide. A model based on the Gouy-Chapman theory in combination with a two-state surface partition equilibrium, separating the electrostatic and the hydrophobic contributions to the binding free energy, was found to be in excellent agreement with the experimental data. Using this model, a surface partition constant of approximately 80 M(-)(1) was obtained for the nonelectrostatic contribution to the binding of penetratin irrespective of the fraction of negatively charged lipids in the membrane, indicating that the hydrophobic interactions are independent of the surface charge density. In accordance with this, circular dichroism measurements showed that the secondary structure of membrane-associated penetratin is independent of the DOPC/DOPG ratio. Experiments using vesicles with entrapped carboxyfluorescein showed that penetratin does not form membrane pores. Studies of the cationic peptide penetratin are complicated by extensive adsorption to surfaces of quartz and plastics. By modification of the quartz cell walls with the cationic polymer poly(ethylenimine), the peptide adsorption was reduced to a tolerable level. The data analysis method used for construction of the binding isotherms eliminated errors emanating from the remaining peptide adsorption, which otherwise would prevent a proper quantification of the binding.     

Inability to Find Consistent Bacterial Biocontrol Agents of Pythium aphanidermatum in Cucumber Using Screens Based on Ecophysiological Traits

A collection of 821 rhizobacteria from cucumber, originating from different root locations and stages of plant development, was screened for potential biocontrol agents of Pythium aphanidermatum (Edson) Fitzp. The screening procedure exploited carbon source utilization profiles and growth rates of bacteria as indicators of a partial niche overlap with the pathogen. The bacteria were tested for growth on nine carbon sources (glucose, fucose, sucrose, maltose, asparagine, alanine, galacturonic acid, succinic acid, and linoleic acid), most of which are reported to be used by the zoospores of P. aphanidermatum in the infection process. The isolates were classified as fast- or slow-growing, depending on their growth rate in 1/10 strength TSB. By nonhierarchical cluster analysis, 20 clusters were generated of bacteria with similar profiles of carbon source utilization. Redundancy analysis showed that the type of root sample explained 47% of the variance found in the relative abundance of bacteria from the clusters. Bacteria from clusters using none or few of the carbon sources, e.g., maltose and linoleic acid, with many slow-growing isolates, showed a preference for plants in the vegetative or generative stage, or for old root regions (root base). Bacteria from clusters with fast-growing isolates, using many carbon sources, were relatively abundant in the seedling stage. A selection of 127 bacteria from the different clusters was tested for disease suppressive capabilities in bioassays on young cucumber plants in nutrient solution, inoculated with zoospores of P. aphanidermatum. Nine of these bacteria produced biosurfactants, and 27 showed antibiosis against mycelial growth in plate assays. For 31 isolates, significant positive effects on plant biomass were shown, as analyzed with a general linear regression model. For most isolates, these effects occurred only in one of two replicate assays and no reductions in the degree of root and crown rot were found. Of the isolates that used many of the tested carbon sources, only four had positive effects on plant biomass. The majority of the isolates that positively affected plant biomass used few to moderate numbers of carbon sources and did not produce antibiotics or biosurfactants. In conclusion, competition for the tested carbon sources with the zoospores did not play a decisive role in disease suppression, and no clear relation was found between ecophysiological traits and disease suppression. Only isolate 3.1T8, isolated from root tips in the generative stage of plant growth, significantly increased plant biomass and suppressed root and crown rot symptoms in five out of six bioassays. The isolate produced an antifungal substance in plate assays and showed biosurfactant production in several (cucumber-derived) media.     

Lysosome mediated Kir2.1 breakdown directly influences inward rectifier current density

The inward rectifier current generated by Kir2.1 ion channel proteins is primarily responsible for the stable resting membrane potential in various excitable cell types, like neurons and myocytes. Tight regulation of Kir2.1 functioning prevents premature action potential formation and ensures optimal repolarization times. While Kir2.1 forward trafficking has been addressed in a number of studies, its degradation pathways are thus far unknown. Using three different lysosomal inhibitors, NH₄Cl, chloroquine and leupeptin, we now demonstrate involvement of the lysosomal degradation pathway in Kir2.1 breakdown. Upon application of the inhibitors, increased steady state protein levels are detectable within few hours coinciding with intracellular granular Kir2.1 accumulation. Treatment for 24 h with either chloroquine or leupeptin results in increased plasmamembrane originating inward rectifier current densities, while current-voltage characteristics remain unaltered. We conclude that the lysosomal degradation pathway contributes to Kir2.1 mediated inward rectifier current regulation.     

Die Kohlehydratverdauung bei Astacus fluviatilis

Zusammenfassung Im Flußkrebsmagensaft fanden wir Amylase, Maltase, sehr wenig In vertase und keine Lactase.Das p_H-Optimum der Amylase ist bei p_H 5,6, der Invertase bei p_H 5,7–6,0. Maltase hat kein scharfes Optimum, sondern ein flaches Optimum zwischen p_H 5 und 6.Die Maltase und die Amylase des Saftes haben ungefähr dieselbe Wirkungsgeschwindigkeit.Die Temperatur verschiebt das p_H-Optimum der Amylase nicht oder nur sehr wenig.Aus der Tatsache, daß die Amylase und die Maltase die gleiche Wirkungsgeschwindigkeit haben, ergibt sich, daß dem Flußkrebs die für die Wirbeltiere charakteristische Regulierung der Ernährung fehlt. Das gleiche dürfte nach Untersuchung von Shinoda und Krueger auch für Eiweißverdauung gelten. Eine Spekulation über die Möglichkeit der Überschwemmung des Krebsorganismus mit Zucker können wir uns sparen, da wir noch zu wenig davon wissen. Nur so viel sei gesagt, daß eine Zuckerresorption im Magen nicht stattfindet, und daß die Menge des Zuckers, die in die Mitteldarmdrüse gelangt, in der Zeiteinheit äußerst gering sein muß, aus Gründen, die wir in der Einleitung besprachen. Schon deswegen muß eine Überschwemmung, wie sie bei den Wirbeltieren stattfinden könnte, hier ausgeschlossen sein. Die Frage, inwieweit überhaupt bei diesen. Tieren eine Regulierung der Kohlehydrate des Blutes lebensnotwendig ist, kann nicht beantwortet werden. Sicherlich aber haben nach Henningsen (11) die Tiere keinen festen Zuckerspiegel, wie ihn die Säugetiere haben, sie können ohne jeden Zuckergehalt des Blutes leben. Nach reichlichen Mahlzeiten steigt die Zuckermenge des Blutes allerdings schnell an, bis etwa 0,030%. Eine Regulation gibt es nur insoweit, als nach Einspritzung größerer Zuckermengen in das Blut der Zuckergehalt des Blutes bald sinkt, bis er gleich demjenigen wird, den man beim gefütterten Tiere findet, solange der Vorrat reicht, wird er dann konstant erhalten. Die große Empfindlichkeit des Säugetieres gegen Schwankungen des Zuckergehaltes im Blute dürfte hier also fehlen. Wie in so vielen Fällen, ist also das niedere Tier Schwankungen der Lebensbedingungen ausgesetzt, es fehlt ihm eine Regulation, wie sie für den Zucker beim Säugetier neben der Leber und den bekannten Hormonen auch durch die beschriebene Eigenartigkeit der Verdauung gewährleistet wird. Wir wollen noch darauf hinweisen, daß Jordan und Begemann (12) gezeigt haben, daß durch den Darm der Schnecken (Helix pomatia) Disaccharide ungefähr ebenso leicht diffundieren, als Monosaccharide (Rohrzucker fast ebenso leicht als Traubenzucker).Dem International Educational sind wir zu großem Dank verpflichtet, da es uns in den Stand gesetzt hat, die benötigte Apparatur anzuschaffen.     

Production of lipid compounds in the yeast Saccharomyces cerevisiae

This review describes progress using the yeast Saccharomyces cerevisiae as a model organism for the fast and efficient analysis of genes and enzyme activities involved in the lipid biosynthetic pathways of several donor organisms. Furthermore, we assess the impact of bakers yeast on the production of novel, high-value lipid compounds. Yeast can be genetically modified to produce selected substances in relatively high amounts. A major advantage in choosing yeast as an object for metabolic engineering is the fact that the lipid pathways in this organism have been described in detail and are well characterized. We focus on the de novo production of three major families of lipid products. These are: (1) sterols, providing some previously known and some novel applications as examples of the lipid pathway enhancement that occurs naturally in yeast, (2) the reconstitution of the biosynthetic pathway of steroid hormones and (3) the biosynthesis of polyunsaturated fatty acids, leading to the biosynthesis of different omega-3 and omega-6 fatty acids which do not occur naturally in yeast. We utilize the current knowledge and point out perspectives and problems for future biotechnological applications in the field of lipid compounds.     

Hydrophobic amino acid residues in the acceptor binding site are main determinants for reaction mechanism and specificity of cyclodextrin-glycosyltransferase

Cyclodextrin-glycosyltransferases (CGTases) (EC ) preferably catalyze transglycosylation reactions with glucosyl residues as acceptor, whereas the homologous alpha-amylases catalyze hydrolysis reactions using water as acceptor. This difference in reaction specificity is most likely caused by the acceptor binding site. To investigate this in detail we altered the acceptor site residues Lys-232, Phe-183, Phe-259, and Glu-264 of Bacillus circulans strain 251 CGTase using site-directed mutagenesis. Lys-232 is of general importance for catalysis, which appears to result mainly from stabilization of the conformation of the loop containing the catalytic nucleophile Asp-229 and His-233, a residue that has been implied in transition state stabilization. Glu-264 contributes to the disproportionation reaction only, where it is involved in initial binding of the (maltose) acceptor. Phe-183 and Phe-259 play important and distinct roles in the transglycosylation reactions catalyzed by CGTase. Mutation of Phe-183 affects especially the cyclization and coupling reactions, whereas Phe-259 is most important for the cyclization and disproportionation reactions. Moreover, the hydrophobisity of Phe-183 and Phe-259 limits the hydrolyzing activity of the enzyme. Hydrolysis can be enhanced by making these residues more polar, which concomitantly results in a lower transglycosylation activity. A double mutant was constructed that yielded an enzyme preferring hydrolysis over cyclization (15:1), whereas the wild type favors cyclization over hydrolysis (90:1).     

Effects of acute and chronic UV-B exposure on a green alga: a continuous culture study using a computer-controlled dynamic light regime

The green alga Selenastrum capricornutum was grown in a specially developed continuous culture system to study long-term effects of chronic UV-B exposure. The new system improves upon previous laboratory culture approaches. It is demonstrated that short-term experiments underestimate UV-B effects. It is also shown that photoinhibition cannot explain the effects under chronic exposure. Under both nutrient-replete and phosphorus-limiting conditions a UV-B mediated delay in cell division rate and an increase in the cellular content of proteins, carbohydrates and chlorophyll a was measured. Transition experiments showed that complete acclimatisation to UV-B took several cell cycles. DNA damage appears to be the major cause of the observed long-term UV-B effects.