Altered velocity processing in schizophrenia during pursuit eye tracking

Smooth pursuit eye movements (SPEM) are needed to keep the retinal image of slowly moving objects within the fovea. Depending on the task, about 50%-80% of patients with schizophrenia have difficulties in maintaining SPEM. We designed a study that comprised different target velocities as well as testing for internal (extraretinal) guidance of SPEM in the absence of a visual target. We applied event-related fMRI by presenting four velocities (5, 10, 15, 20°/s) both with and without intervals of target blanking. 17 patients and 16 healthy participants were included. Eye movements were registered during scanning sessions. Statistical analysis included mixed ANOVAs and regression analyses of the target velocity on the Blood Oxygen Level Dependency (BOLD) signal. The main effect group and the interaction of velocity×group revealed reduced activation in V5 and putamen but increased activation of cerebellar regions in patients. Regression analysis showed that activation in supplementary eye field, putamen, and cerebellum was not correlated to target velocity in patients in contrast to controls. Furthermore, activation in V5 and in intraparietal sulcus (putative LIP) bilaterally was less strongly correlated to target velocity in patients than controls. Altered correlation of target velocity and neural activation in the cortical network supporting SPEM (V5, SEF, LIP, putamen) implies impaired transformation of the visual motion signal into an adequate motor command in patients. Cerebellar regions seem to be involved in compensatory mechanisms although cerebellar activity in patients was not related to target velocity.     

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Förster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo^1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest^3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells⁵, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium⁶, cAMP^7-8, inositol phosphates⁹ and cGMP^10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a β-adrenergic receptor agonist and blocker.     

Aggressive females become aggressive males in a sex-changing reef fish

Many animal populations display consistent individual differences in suites of correlated behaviours. While these so called 'animal personalities' can substantially influence the ecology and evolution of populations, little is known about cross-sex correlations of behaviour and thus the potential of personality to limit sex-specific behavioural adaptations. Here, we experimentally induced sex-change in the sequentially hermaphroditic reef fish Parapercis cylindrica and demonstrate the existence of tight cross-sex correlations for two behaviours with presumed different sex-specific optima. Individuals that were relatively more active and aggressive females were found to become relatively more active and aggressive males. By identifying personality as a potential genetic constraint on the resolution of intralocus sexual conflict over behaviour, our findings have important ecological and evolutionary implications for a wide range of species.     

Identification of catalytically important residues in the active site of Escherichia coli transaldolase.

The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site-directed mutagenesis. The mutant enzymes D17A, N35A, E96A, T156A, and S176A were purified from a talB-deficient host and analyzed with respect to their 3D structure and kinetic behavior. X-ray analysis showed that side chain replacement did not induce unanticipated structural changes in the mutant enzymes. Three mutations, N35A, E96A, and T156A resulted mainly in an effect on apparent kcat, with little changes in apparent Km values for the substrates. Residues N35 and T156 are involved in the positioning of a catalytic water molecule at the active site and the side chain of E96 participates in concert with this water molecule in proton transfer during catalysis. Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity. The apparent Km value for the donor substrate, fructose 6-phosphate, was increased nearly fivefold while the apparent Km value for the acceptor substrate, erythrose 4-phosphate remained unchanged, consistent with a function for S176 in the binding of the C1 hydroxyl group of the donor substrate. The mutant D17A showed a 300-fold decrease in kcat, and a fivefold increase in the apparent Km value for the acceptor substrate erythrose 4-phosphate, suggesting a role of this residue in carbon-carbon bond cleavage and stabilization of the carbanion/enamine intermediate.     

The efficient export of NADP-containing glucose-fructose oxidoreductase to the periplasm of Zymomonas mobilis depends both on an intact twin-arginine motif in the signal peptide and on the generation of a structural export signal induced by cofactor binding.

The periplasmic, NADP-containing glucose-fructose oxidoreductase of the gram-negative bacterium Zymomonas mobilis belongs to a class of redox cofactor-dependent enzymes which are exported with the aid of a signal peptide containing a so-called twin-arginine motif. In this paper we show that the replacement of one or both arginine residues results in drastically reduced translocation of glucose-fructose oxidoreductase to the periplasm, showing that this motif is essential. Mutant proteins which, in contrast to wild-type glucose-fructose oxidoreductase, bind NADP in a looser and dissociable manner, were severely affected in the kinetics of plasma membrane translocation. These results strongly suggest that the translocation of glucose-fructose oxidoreductase into the periplasm uses a Sec-independent apparatus which recognizes, as an additional signal, a conformational change in the structure of the protein, most likely triggered by cofactor binding. Furthermore, these results suggest that glucose-fructose oxidoreductase is exported in a folded form. A glucose-fructose oxidoreductase:beta-galactosidase fusion protein is not lethal to Z. mobilis cells and leads to the accumulation of the cytosolic preform of wild-type glucose-fructose oxidoreductase expressed in trans but not of a typical Sec-substrate (OmpA), indicating that the glucose-fructose oxidoreductase translocation apparatus can be blocked without interfering with the export of essential proteins via the Sec pathway.     

Durchflußfluorescenzcytophotometrie für ultraschnelle DNS-Messungen an großen Zellpopulationen

Zusammenfassung Ausgehend vom Leitz-Mikroskopphotometer MPV mit Auflicht-Anregung wird ein Durchflußfluorescenzcytophotometer beschrieben, das es gestattet, die relativen Feulgen-DNS-Mengen von bis zu 1000 Zellen pro Sekunde zu bestimmen. Die Meßwerte werden in einem Impulshöhenanalysator klassifiziert und gespeichert. Auf Abruf wird der Speicherinhalt analog als Histogramm ausgeschrieben oder digital als Zahlenfolge ausgedruckt.Das Vorgehen bei der Zellsuspensionsherstellung, Fixierung, Hydrolyse und Färbung mit dem Feulgen-Farbstoff Akriflavin wird dargelegt.Anwendungsbeispiele werden besprochen.

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Flow-through-cytophotometry for ultra-rapid DNA measurement of large cell populations

Summary A flow-through cytophotometer based upon the Leitz-microscope photometer (MPV) is described. The light source is a high pressure Xenon arc HBO 100 in an arrangement for epiillumination.The flow system is designed in such a way that each cell, after previous Feulgenstaining in aqueous suspension, has to pass through the focal plane of the objective. This is achieved by a self-constructed flow channel with a vertical fluid stram carrying the stained cells along the optical axis of the microscope at a flow rate of 100 to 1000 cells per sec. After passage through the focal plane with simultanneous registration of the highest fluorescence reading the cells are flushed off by a horizontal water current.The fluorescence light impulses emitted by the cells are classified and stored by an impulse height discriminator, from which they may be recalled either analogically as a histogram (DNA distribution pattern) or digitally as a series of numbers.In addition, the fluorescence impulses can be visualized at the screen of an oscillograph. The flow rate of the fluorochromized cells is determined by means of a counter unit.The steps of preparation of the cell suspension as well as fixation, hydrolysis and Feulgen-staining with acriflavine-Schiff of the suspended tissue cells is described in detail.The possibilities of application of the instrument as a cyto-morphometric approach to cytology and cytochemistry automation are discussed.

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Mit Unterstützung des Bundesministeriums für Jugend, Familie und Gesundheit.

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Zu besonderem Dank sind wir der Fa. Leitz, Wetzlar, verpflichtet. Sie stellte kostenlos das Mikroskopphotometer mit Lichtmeßeinrichtung zur Verfügung und ermöglichte uns die Überprüfung der Apparatur in ihren Laboratorien. Den wissenschaftlichen Mitarbeitern der Fa. Leitz, Herrn Ing. J. Rzeznik und Herrn Dipl. Phys. H. Wasmund, danken wir für ihre Beratung und die anregende Diskussion des Manuskriptes.Herrn Ing. H. Eipel, Fa. Elektronik Service, Frankfurt, sei für die Beratung bei Problemen der Impulsanalyse und Durchsicht des Manuskriptes gedankt.Ebenfalls danken wir Fräulein M. Schaden für die gewissenhafte und selbständige Durchführung der biologischen Versuche.     

Torso receptor activity is regulated by a diffusible ligand produced at the extracellular terminal regions of the Drosophila egg.

torso encodes a receptor tyrosine kinase (torso) required for anterior and posterior terminal development of the Drosophila embryo. Injecting eggs with in vitro synthesized torso mRNAs revealed that torso activation is governed by an extracellular molecule, presumably the torso ligand, produced at terminal regions of the egg during early embryogenesis. In the absence of torso, the ligand shows no apparent localization, indicating that it is diffusible and normally bound by an excess of torso receptor at the egg poles. Mutant ligand-binding torso proteins can suppress telson formation in a dominant negative manner, suggesting that the ligand is limited in amount. Analysis of torso mutations indicates that torso functions as a tyrosine kinase and that gain-of-function mutations causing ligand-independent activation are located in the extracellular domain.     

Continuous production of erythrulose using transketolase in a membrane reactor

Transketolase can be used for synthesis of chiral intermediates and carbohydrates. However the enzyme is strongly deactivated by the educts. This deactivation depends on the reactor employed. An enzyme membrane reactor allows the continuous production of L-erythrulose with high conversion and stable operational points. A productivity (space-time yield) of 45g L d was reached.